CA3214538A1

CA3214538A1 - Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof

Info

Publication number: CA3214538A1
Application number: CA3214538A
Authority: CA
Inventors: Joel DE BEER; Monique MAURER; Nicolas Meier; Lavaniya KUNALINGAM; Marcello CLERICI
Original assignee: Anjarium Biosciences AG
Current assignee: Anjarium Biosciences AG
Priority date: 2021-04-20
Filing date: 2022-04-19
Publication date: 2022-10-27
Also published as: EP4326860A1; WO2022223556A1; AU2022260111A1; KR20240012370A; AU2022260111A9

Abstract

Provided herein are double strand DNA molecules comprising inverted repeats, expression cassette and one or more restriction sites for nicking endonucleases, the methods of use thereof, and the methods of making therefor.

Description

COMPOSITIONS OF DNA MOLECULES ENCODING AMYLO-ALPHA-1, 6-GLUCOSIDASE, 4-ALPHA-GLUCANOTRANSFERASE, METHODS OF MAKING THEREOF, AND METHODS OF USE
THEREOF
PRIORITY
[0001] This application claims the benefit of priority to U.S.
Serial No. 63/177,016 filed April 20, 2021, which is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled "14497-008-228 Sequence Listing.txt" created on April 19, 2022 and having a size of 167,403 bytes.
1. Field

[0003] Provided herein are double strand DNA molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, the methods of use thereof, and the methods of making thereof Also provided are methods of treating glycogen storage disorders.
2. Background

[0004] Gene therapy aims to introduce genes into target cells to treat or prevent disease.
By supplying a transcription cassette with an active gene product (sometimes referred to as a transgene), the application of gene therapy can improve clinical outcomes, as the gene product can result in a gain of positive function effect, a loss of negative function effect, or another outcome, such as in patients suffering from cancer, can have an oncolytic effect.
Delivery and expression of a corrective gene in the patient's target cells can be carried out via numerous methods, including non-viral delivery (e.g. liposomal) or viral delivery methods that include the use engineered viruses and viral gene delivery vectors. Among the available virus-derived vectors, also known as viral particles, (e.g., recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, and the like), AAV systems are gaining popularity as a versatile vector in gene therapy.

[0005] However, there are several major deficiencies in using viral particles as a gene delivery vector. One major drawback is the dependency on viral life cycle and viral proteins to package the transcription cassette into the viral particles. As a result, use of viral vectors

6 has been limited in terms of size of transgenes (e.g. less than 150,000 Da protein coding capacity for AAV) or the requirement for specific viral sequences to be present to ensure efficient replication and packaging (e.g. Rep-Binding Element), which can in turn destabilize the expression cassette. Thus, more than one viral particle may be required to deliver large transgenes (e.g., transgenes encoding proteins larger than 150,000 Da, or transgenes longer than about 4.7 Kb). Use of two or more AAV constructs can increase the risk of re-activation of the AAV genome.
100061 The second drawback is that viral particles used for gene therapy are often derived from wild-type viruses to which a subset of the population has been exposed during their lifetime. These patients are found to carry neutralizing antibodies which can in turn hinder gene therapy efficacy as further described in Snyder, Richard 0., and Philippe Moullier.
Adeno-associated virus : methods' and protocols. Totowa, NJ: Humana Press, 2011. Print...
For the remaining seronegative patients, the capsids of viral vectors are often immunogenic, preventing re-administration of the viral vector therapy to patients should an initial dose not be sufficient or should the therapy wear off.
100071 As such, there is unmet need for non-viral-based gene therapies as an alternative to viral particles, particularly therapies that delivery large transgenes.
Additionally, there is unmet need for methods to produce these capsid free vectors in host cells without the co-presences of a plasmid or DNA sequences that encode for the viral replication machinery (e.g. AAV Rep genes), because these viral proteins or the viral DNA sequences encoding for them can contaminate the isolated DNA of a capsid free viral vector.
100081 Furthermore, there remains an important unmet need for recombinant DNA
vectors with improved production and/or expression properties. There is also an unmet need for non-immunogenic gene delivery vectors that allow for repeat administration without loss of efficacy due to, e.g., neutralizing antibodies.
100091 Disorders related to impaired or missing function of amyl o-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE), including glycogen storage diseases GSDIII
types A-C , cause defects in glycogen metabolism. Specifically, the debranching activity of GDE is impaired, leading to accumulation of glycogen in different tissues, with the liver being most affected. Due to the metabolic defects, patients suffer from low blood sugar (hypoglycemia), enlargement of the liver (hepatomegaly), excessive amounts of fat in the blood (hyperlipidemia), elevated blood levels of liver enzymes, chronic liver disease (cirrhosis), liver failure, slow growth, short stature, benign tumors (adenomas), hypertrophic cardiomyopathy, cardiac dysfunction, congestive heart failure, skeletal myopathy, and/or poor muscle tone (hypotonia). Currently, disease management is limited to dietary treatment to preventing severe ketotic hypoglycemia at very young ages. The strict diet must begin as soon as possible after birth and be continued for at least 15 years, if not lifelong.
Furthermore, most of the GSDIII patients develop long-term pathologies.
Despite recent successes with adeno-associated virus (AAV)-based gene replacement for metabolic diseases, current limitations of AAV-mediated gene transfer still represent a challenge for successful gene therapy in GSDIII, including the size of the gene (Louisa Jauze et al.
Human Gene Therapy; Oct 2019.1263-1273). Furthermore, loss of transgene over time has been observed in liver directed AAV gene therapies, possibly due to the pathological state of the to be treated hepatocytes.
[0010] Despite the great advances in understanding the molecular biology, and diagnosis of GSDIII, little progress has been made in developing new treatments for the disorder. There remains large unmet need for durable disease-modifying therapies in GSDIII The current therapies are mainly aimed at short term maintained of normoglycemia, that require strict dietary restrictions, and non-compliance can lead to seizures and in extreme cases coma.
Furthermore the need to prevent long term damage to tissues such as the liver (including severe fibrosis) and muscles remains unaddressed. There are no approved gene therapies for GSDIII, and regular AAV based therapies cannot accommodate the large transgene nor can they be used by 25% to 40% of patients due to pre-existing antibodies. Other viral gene therapy vectors that may accommodate the large transgene pose the challenge that they can only be administered once, and the resulting GDE expression levels might not be high enough to be efficacious, or may be supranormal dose levels cannot be titrated.
[0011] Accordingly, there is need in the field for a technology that permits expression of a therapeutic GDE protein in a cell, tissue or subject for the treatment of GDSIII.
3. Summary 100121 Provided herein is a method for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a human patient, the method comprising administering to the patient a biocompatible carrier (hybridosome) or lipid nanoparticle, wherein the hybridosome or the lipid nanoparticle comprises a DNA
molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof.

100131 Provided herein is a method for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a human patient, the method comprising administering to the patient a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, wherein the DNA molecule is contained within a single delivery vector.
100141 Provided herein is a method for treating a disease associated with reduced activity of GDE in a human patient, the method comprising the steps of (i) administering a first dose of a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof to the patient and (ii) administering a second dose of the DNA molecule to the patient.
100151 In one embodiment, the first dose of the DNA molecule is administered to the patient at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months before the second dose of the DNA molecule.
100161 In one embodiment, the first dose of the DNA molecule is administered to the patient at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least years, at least 15 years, or at least 20 years before the second dose of the DNA molecule.
100171 In one embodiment, the first dose of the double-stranded DNA
molecule and the second dose of the DNA molecule contain the same amount of the DNA molecule.
100181 In one embodiment, the first dose of the DNA molecule and the second dose of the DNA molecule contain different amounts of the DNA molecule.
100191 In one embodiment, the method further comprises administering one or more additional doses of the DNA molecule.
100201 In one embodiment, the DNA molecule is administered once weekly, biweekly, or monthly.
100211 In one embodiment, the DNA molecule is administered to the patient about every 6 months, about every 12 months, about every 18 months, about every 2 years, about every 3 years, about every 5 years, about every 10 years, about every 15 years or about every 20 years 100221 In one embodiment, the DNA molecule is administered to the patient for the duration of the life of the patient.
100231 In one embodiment, the patient is an adult patient.
100241 In one embodiment, the patient is a pediatric patient.

100251 In one embodiment, the patient is a pediatric patient when the first dose of the DNA molecule is administered.
100261 In one embodiment, the pediatric patient is an infant.
100271 In one embodiment, the pediatric patient is about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, or about 18 years old.
100281 In one embodiment, the disease is Glycogen Storage Disease (GDS) Type III
(GSDIII).
100291 In one embodiment, the disease is GSDIIIa, GSDIIIb, GSDIIIc, and GSDIIId.
100301 In one embodiment, the transgene comprises a sequence that is at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence set forth in SEQ ID NO:
174, 175, 178, or 179 100311 In one embodiment, the method results in an improvement of one or more of the following clinical symptoms of GSDIII: fasting intolerance, exercise intolerance, growth failure, myopathy, muscle weakness, and hepatomegaly.
100321 In one embodiment, the method results in a reduction in the number of hypoglycemic episodes per year of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%
in the patient.
100331 In one embodiment, the method results in an improvement in liver function of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% in a patient as determined by liver function tests.
100341 In one embodiment, the method results in a reduction in the number of hyperlipidemic episodes per year of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% in the patient.
100351 In one embodiment, the method results in a clinical improvement of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater than about 95% as measured by one or more of the following metabolic markers: glucose, lactate, ketones, creatine phosphokinase, uric acid, lipids or ketones.
100361 In one embodiment, the method results in a clinical improvement of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater than about 95% as measured by the levels of urinary glucose tetrasaccharide (G1c4) in the patient.
100371 In one embodiment, the method results in GDE protein activity of about 1-10%, about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, or about 80-90% of the biological activity level of the native GDE protein.
100381 In one embodiment, the DNA molecule is detectable in the hepatocytes of the patient by quantitative real-time PCR.
100391 In one embodiment, the method results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 95% decrease in limit dextrin accumulation in a biological sample (e.g., a liver sample) from the patient.
100401 In one embodiment, the DNA molecule is detectable in the muscle tissue of the patient by quantitative real-time PCR.
100411 In one embodiment, the method results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 95% decrease in limit dextrin accumulation in a biological sample (e.g., a muscle sample) from the patient.
100421 Provided herein is a double-stranded DNA molecule comprising in 5' to 3' direction of the top strand:
(a) a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
(b) an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof; and (c) a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.
100431 Provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand:

(a) a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
(b) an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof; and (c) a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.
100441 Provided herein is a double-stranded DNA molecule comprising in 5' to 3' direction of the top strand (a) a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
(b) an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof; and (c) a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.
100451 Provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand:
(a) a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
(b) an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof; and (c) a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted

7 repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.
[0046] In one embodiment, the DNA molecule provided herein is an isolated DNA
molecule.
[0047] In one embodiment, the first, second, third, and fourth restriction sites for nicking endonuclease of a DNA molecule provided herein are all restriction sites for the same nicking endonuclease.
[0048] In one embodiment, the first and the second inverted repeats of a DNA molecule provided herein are the same.
[0049] In one embodiment, the first and/or the second inverted repeat of a DNA molecule provided herein is an ITR of a parvovirus.
[0050] In one embodiment, the first and/or the second inverted repeat of a DNA molecule provided herein is a modified ITR of a parvovirus [0051] In one embodiment, the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus.
[0052] In one embodiment, the nucleotide sequence of the modified ITR of a DNA
molecule provided herein is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99%
identical to the ITR of the parvovirus.
[0053] In one embodiment, the ITR of a DNA molecule provided herein comprises a viral replication-associated protein binding sequence ("RABS").
[0054] In one embodiment, the RABS comprises a Rep binding sequence.
[0055] In one embodiment, the RABS comprises an NS1-binding sequence.
[0056] In one embodiment, the ITR of a DNA molecule provided herein does not comprise a RABS.
[0057] In one embodiment, the transgene comprises a sequence of SEQ
ID NO: 174, 175, 178, or 179.
[0058] In one embodiment, a DNA molecule provided herein is such that:
(a) the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
(b) the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;

8 (c) the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat; and/or (d) the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat.
100591 In one embodiment, a DNA molecule provided herein is such that:
(a) the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
(b) the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
(c) the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat; and/or (d) the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat.
100601 In some embodiment, a DNA molecule provided herein is such that:
(a) the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
(b) the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
(c) the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat; and/or (d) the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat.
100611 In some embodiment, a DNA molecule provided herein is such that:

9 (a) the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
(b) the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
(c) the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat; and/or (d) the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat 100621 In one embodiment, the nick is inside the inverted repeat 100631 In one embodiment, the nick is outside the inverted repeat.
100641 In one embodiment, the DNA molecule is a plasmid.
100651 In one embodiment, the plasmid further comprises a bacterial origin of replication.
100661 In one embodiment, the plasmid further comprises a restriction enzyme site in the region 5' to the first inverted repeat and 3' to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and the region between the first and second inverted repeats.
100671 In one embodiment, the cleavage with the restriction enzyme results in single strand overhangs that do not anneal at detectable levels under conditions that favor annealing of the first and/or second inverted repeat.
100681 In one embodiment, the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are.
(a) on opposite strands; and (b) create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat.
100691 In one embodiment, the fifth and the sixth nick are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart.
100701 In one embodiment, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are all target sequences for the same nicking endonuclease.

100711 In one embodiment, the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is Nt. BsmAI;
Nt. BtsCI; N.
ALw1; N. BstNBI; N. BspD6I; Nb. Mva12691; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt.
Bpul0I; Nt.
BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI.
100721 In one embodiment, the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALw1; N.
BstNBI; N.
BspD6I; Nb. Mval2691; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpul0I; Nt. BsmBI;
Nb. BbvCI;
Nt. BbvCI; or Nt. BspQI.
100731 In one embodiment, the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is a programmable nicking endonuclease.
100741 In one embodiment, the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease is a programmable nicking endonuclease 100751 In one embodiment, the nicking endonuclease is a Cas nuclease.
100761 In one embodiment, the expression cassette further comprises a promoter operatively linked to a transcription unit.
100771 In one embodiment, the transcription unit comprises an open reading frame.
100781 In one embodiment, the expression cassette further comprises a posttranscriptional regulatory element.
100791 In one embodiment, the expression cassette further comprises a polyadenylation and termination signal.
100801 In one embodiment, the size of the expression cassette is at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb.
100811 Provided herein is a kit for expressing a human GDE in vivo, the kit comprising 0.1 to 500 mg of a DNA molecule provided herein and a device for administering the DNA
molecule.
100821 In one embodiment, the device is an injection needle.
100831 Provided herein is a composition comprising one or more DNA
molecules provided herein, and a pharmaceutically acceptable carrier.
100841 In one embodiment, the carrier comprises a transfection reagent, a nanoparticle, a hybridosome, or a liposome.
100851 In one embodiment, a composition provided herein is used in medical therapy.

100861 In one embodiment, a composition provided herein is used for preparing or manufacturing a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of GDE in a subject need thereof.
4. Brief Description of the Drawings 100871 FIG. 1 depicts the structures of various exemplary hairpins and the structural elements of the hairpins.
100881 FIGS. 2A-2C depict a linear interaction plot showing exemplary strand conformations and intramolecular forces within the overhang as well as intermolecular forces between the strands. FIG 2C depicts the expected annealed structure of FIG 2A
and FIG 2B.
100891 FIGS. 3A-3C depict various exemplary arrangements of hairpins and the location of various restriction sites as well as restriction sites for type II nicking endonucleases in the primary stem of a hairpin 100901 FIG. 4 depicts the structures of various exemplary hairpins and the structural elements of human mitochondrial DNA OriL and OriL derived ITRs.
100911 FIG. 5 depicts the structures of hairpins of an exemplary aptamer and aptamer ITR.
100921 FIG. 6A illustrates an exemplary structure of a circular plasmid from which DNA
products for the expression of an GDE protein as disclosed herein, arise after performing method steps as described in Example L
100931 FIG. 6B illustrates an exemplary structure of a hairpin-ended DNA molecule for the expression of a GDE protein as disclosed herein. In this embodiment, the exemplary hairpin-ended DNA comprises an expression cassette containing a PGK promoter, an open reading frame (ORF) encoding the GDE transgene and BGH poly(A) tail. The expression cassette is flanked by two single stranded terminal hairpins. FIG. 6C depicts a visualization of DNA products from construct 1 after performing method steps as described in Example 1.
100941 FIG. 7A illustrates a further exemplary structure of a plasmid from which DNA
products for the expression of an GDE protein as disclosed herein, arise after performing method steps as described in Example 1. In this embodiment, twelve (six doubles) restriction sites for nicking endonuclease (e.g. restriction site for nicking endonuclease as described in Sections 5.3.4 and 5.4.2) in the region 5' to the first inverted repeat and 3' to the second inverted repeat.
100951 FIG. 7B illustrates an exemplary structure of a hairpin-ended DNA molecule for the expression of a GDE protein as disclosed herein. In this embodiment, the exemplary hairpin-ended DNA comprises an expression cassette containing promoter, an open reading frame (ORF) encoding the GDE transgene, a WPRE regulatory element, and a poly(A) tail.
The expression cassette is flanked by two single stranded terminal hairpins.
Unique restriction endonuclease recognition sites were also introduced between each component to facilitate the introduction of new genetic components into the specific sites in the construct.
100961 FIGS. 8A and 8B show GDE protein activity of cells transfected with hairpin-ended DNA molecules encoding GDE.
100971 FIG. 9A depicts the glycogen content converted to glucose in the lysate of glucose starved GSDIII patient derived fibroblasts treated with hairpin-ended DNA
molecules encoding GDE or GFP, over time. FIG. 9B depicts the glycogen content converted to glucose in the lysate of glucose starved wild type GDE expressing fibroblasts treated with hairpin-ended DNA molecules encoding GDE or GFP, over time.
100981 FIGS. 10A-10C depict luciferase expression in dividing and non-dividing cells as described in Section 6.3. FIG. 10A depicts expression over time of luciferase by non-dividing transfected with equimolar amounts of hairpin-ended DNA molecules encoding a secreted luciferase encapsulated in LNPs or Hybridosomes. FIG. 10B depicts expression of luciferase following transfection equimolar amounts of hairpin-ended DNA
molecules and full circular plasmid each encoding the identical expression cassette for secreted luciferase, encapsulated in hybridosomes by non-dividing cells. FIG. 10C depicts expression of luciferase following transfection equimolar amounts of hairpin-ended DNA
molecules and full circular plasmid encoding the identical expression cassette for secreted luciferase encapsulated in hybridosomes by dividing cells. Luciferase activity peaks in dividing cells on day 2, while in non-dividing cells the expression continues for 4 weeks. In non-dividing cells, as a direct comparison, the luciferase expression by the full circular plasmid diminishes over time.
100991 FIG. 11 depicts a sequence alignment of ITRs derived from AAV1 highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.
1001001 FIG. 12 depicts a sequence alignment of ITRs derived from AAV2 highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.
1001011 FIG. 13 depicts a sequence alignment of ITRs derived from AAV3 highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.

1001021 FIG. 14 depicts a sequence alignment of ITRs derived from AAV4 Left highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.
1001031 FIG. 15 depicts a sequence alignment of ITRs derived from AAV4 Right highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.
1001041 FIG. 16 depicts a sequence alignment of ITRs derived from AAV5 highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites.
1001051 FIG. 17 depicts a sequence alignment of ITRs derived from AAV7 highlighting sequence modifications to generate recognition sites for different nicking endonucleases recognition sites 5. Detailed Description 1001061 Provided herein are methods and compositions for the treatment of a disease or disorder associated with reduced presence or function of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a subject. In some embodiments, the disease associated with reduced presence or function of GDE is Glycogen Storage Disease Type III
(GSDIII).
Such compositions include a hairpin-ended DNA molecule, comprising one or more nucleic acids that encode an GDE therapeutic protein or fragment thereof In one embodiment, a composition described herein includes a hairpin-ended DNA molecule comprising one nucleic acid that encode an GDE therapeutic protein or fragment thereof. In one embodiment, a composition described herein includes a hairpin-ended DNA
molecule comprising two, three, four, or more nucleic acids that encode an GDE
therapeutic protein or fragment thereof Also provided herein are hairpin-ended DNA molecules for the expression of the GDE protein as described herein comprising one or more nucleic acids that encode for the GDE protein. Also provided herein are methods of manufacturing hairpin-ended DNA
molecules described herein. Also provided herein are methods of treating GSDIII using the hairpin-ended DNA provided herein and related pharmaceutical compositions.
More specifically, provided herein are methods of treating GSDIII comprising administering to a subject in need thereof the hairpin-ended DNA described herein.
1001071 Provided herein are methods of making hairpin-ended DNA molecules.
Also provided herein are methods of using hairpin-ended DNA molecules, including for example, using hairpin-ended DNA molecules for gene therapies. The various methods of making the hairpin-ended DNA molecules are further described in Section 5.2 below. The various methods of using hairpin-ended DNA molecules are described in Section 5.8 below. The hairpin-ended DNA made by these methods are provided in Section 5.5 below and include hairpinned inverted repeats at the two ends and an expression cassette, each of which are further described below. In some embodiments, the hairpin-ended DNA also include one or two nicks, as further provided below in Section 5.5 below. Hairpin, hairpinned inverted repeats, and the hairpinned ends are described in Section 5.5 below; the inverted repeats that form the hairpinned ends are described in Section 5.4.1 below; the nicks, nicking endonuclease, and restriction sites for nicking endonuclease are described in Sections 5.4.2 and 5.5 below; the expression cassette are described in Sections 5.4.3 and 5.5 below; and the functional properties of the hairpin-ended DNA molecules are described in Section 5.6 below. As such, the disclosure provides hairpin-ended DNA molecules, methods of making thereof, methods of using therefor, with any combination or permutation of the components provided herein.
1001081 Also provided herein are parent DNA molecules used in the methods to make the hairpin-ended DNA molecules, which parent DNA molecules include two inverted repeats, two or more restriction sites for nicking endonuclease, and an expression cassette, each of which are further described below. The restriction sites for nicking endonuclease are arranged such that, upon nicking by the nicking endonuclease and denaturing, single strand overhangs with inverted repeat sequences form, which then fold to form hairpins upon annealing, each step as described in Section 5.2. The inverted repeats are described in Section 5.4.1 below; the nicks, nicking endonuclease, and restriction sites for nicking endonuclease are described in Section 5.4.2 below; the expression cassette are described in Section 5.4.3 below. As such, the disclosure provides parent DNA molecules used in the methods of making, with any combination or permutation of the components provided herein.
5.1 Definitions 1001091 As used herein, the term "isolated" when used in reference to a DNA
molecule is intended to mean that the referenced DNA molecule is free of at least one component as it is found in its natural, native, or synthetic environment. The term includes a DNA molecule that is removed from some or all other components as it is found in its natural, native, or synthetic environment. Components of a DNA molecule's natural, native, or synthetic environment include anything in natural native, or synthetic environment that are required for, are used in, or otherwise play a role in the replication and maintenance of the DNA

molecule in that environment. Components of a DNA molecule's natural, native, or synthetic environment also include, for example, cells, cell debris, cell organelles, proteins, peptides, amino acids, lipids, polysaccharides, nucleic acids other than the referenced DNA molecule, salts, nutrients for cell culture, and/or chemicals used for DNA synthesis. A
DNA molecule of the disclosure can be partly, completely, or substantially free from all of these components or any other components of its natural, native, or synthetic environment from which it is isolated, synthetically produced, naturally produced, or recombinantly produced. Specific examples of isolated DNA molecules include partially pure DNA molecules and substantially pure DNA molecules.
[00110] As used herein, the term "delivery vehicle" refers to substance that can be used to administer or deliver one or more agents to a cell, a tissue, or a subject, particular a human subject, with or without the agent(s) to be delivered. A delivery vehicle may preferentially deliver agent(s) to a particular subset or a particular type of cells The selective or preferential delivery achieved by the delivery vehicle can be achieved the properties of the vehicle or by a moiety conjugated to, associated with, or contained in the delivery vehicle, which moiety specifically or preferentially binds to a particular subset of cells. A delivery vehicle can also increase the in vivo half-life of the agent to be delivered, the efficiency of the delivery of the agent comparing to the delivery without using the delivery vehicle, and/or the bioavailability of the agent to be delivered. Non-limiting examples of a delivery vehicle are hydridosomes, liposomes, lipid nanoparticles, polymersomes, mixtures of natural/synthetic lipids, membrane or lipid extracts, exosomes, viral particles, protein or protein complexes, peptides, and/or polysaccharides.
[00111] As used herein, the term "subject" refers to a human or any non-human animal (e.g. , mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A
subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. In an exemplary embodiment, a subject of the present disclosure is a subject with reduced activity (e.g., resulting from reduced concentration, presence, and/or function) of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL). In a further exemplary embodiment, the subject is a human.
1001121 The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B, A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C;
A (alone); B (alone); and C (alone).
5.2 Hairpin-ended DNA Molecules and Methods of Making the Hairpin-ended DNA Molecules [00113] The methods and compositions described herein involve compositions and methods for delivering a GDE nucleic acid sequence encoding human GDE protein to subjects in need thereof for the treatment of GSDIII.
[00114] In some embodiments, polynucleotide molecules for expressing a human amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (collectively or individually referred to herein as "AGL" or "GDE") or a fragment thereof having GDE activity.
[00115] In some embodiments, the hairpin-ended DNA molecules of this disclosure can be used in methods for ameliorating, preventing or treating one or more of GSDIIIa, GSDIIIb, GSDIIIc, and GSDIIId (collectively or individually referred to herein as "GSDIII"
or "glycogen storage disease type III").
1001161 The disease or disorder to be treated herein (e.g. , GSDIIIa, GSDIIIb, GSDIIIc, or GSDIIId) may be associated with low blood sugar (hypoglycemia), enlargement of the liver (hepatomegaly), excessive amounts of fat in the blood (hyperlipidemia), elevated blood levels of liver enzymes, chronic liver disease (cirrhosis), liver failure, slow growth, short stature, benign tumors (adenomas), hypertrophic cardiomyopathy, cardiac dysfunction, congestive heart failure, skeletal myopathy, and/or poor muscle tone (hypotonia).
[00117] As is understood by the skilled artisan, GSDIII may be referred to by any number of alternative names in the art, including, but not limited to, AGL
deficiency, Cori disease, Con's disease, debrancher deficiency, Forbes disease, glycogen debrancher deficiency, GSDIII, or limit dextrinosis Accordingly, GSDIII may be used interchangeably with any of these alternative names in the specification, the examples, the drawings, and the claims.
[00118] In a further aspect, provided herein are methods for making a preparing a hairpin-ended DNA molecule for expressing a human amyl o-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL). In one aspect, provided herein is a method for preparing a hairpin-ended DNA molecule, wherein the method comprises: a. culturing a host cell comprising the DNA molecule as described in Section 5.4 under conditions resulting in amplification of the DNA molecule; b. releasing the DNA molecule from the host cell; c. incubating the DNA
molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks; d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;
e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d.
5.3 Methods of Making the Hairpin-ended DNA Molecules 1001191 In one aspect, provided herein is a method for preparing a hairpin-ended DNA
molecule, wherein the method comprises: a. culturing a host cell comprising the DNA
molecule as described in Section 5.4 under conditions resulting in amplification of the DNA
molecule; b. releasing the DNA molecule from the host cell; c. incubating the DNA
molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks; d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;
e annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d.
1001201 In another aspect, provided herein is a method for preparing a hairpin-ended DNA, wherein the method comprises: a. culturing a host cell comprising the plasmid of 5.4.6 under conditions resulting in amplification of the plasmid; b. releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks; d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs; e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA
fragment resulting from step d; f. incubating the plasmid or the fragments resulting from step d with the restriction enzyme and thereby cleaving the plasmid or a fragment of the plasmid;
and g. incubating the fragments of the plasmid with an exonuclease thereby digesting the fragments of the plasmid except the fragment resulting from step e.
1001211 In a further aspect, provided herein is a method for preparing a hairpin-ended DNA, wherein the method comprises: a. culturing a host cell comprising the plasmid of claim 24 under conditions resulting in amplification of the plasmid; b.
releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more nicking endonuclease recognizing the first, second, third, and fourth restriction sites resulting in four nicks; d.
denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs; e. annealing the single strand DNA

overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d; f. incubating the plasmid or the fragments resulting from step d with one or more nicking endonuclease recognizing the fifth and sixth restriction sites resulting in the break in the double stranded DNA molecule; and g.
incubating the fragments of the plasmid with an exonuclease thereby digesting the fragments of the plasmid except the fragment resulting from step e.
1001221 In one aspect, provided herein is a method for preparing a hairpin-ended DNA
molecule, wherein the method comprises: a. culturing a host cell comprising the DNA
molecule as described in Section 5.4 under conditions resulting in amplification of the DNA
molecule; b. releasing the DNA molecule from the host cell; c. incubating the DNA
molecule with one or more programmable nicking enzyme recognizing the four target sites for the guide nucleic acid resulting in four nicks; d. denaturing and thereby creating a DNA
fragment that comprises the expression cassette and is flanked by the two single strand DNA
overhangs; e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d.
1001231 In another aspect, provided herein is a method for preparing a hairpin-ended DNA, wherein the method comprises: a. culturing a host cell comprising the plasmid of 5.4.6 under conditions resulting in amplification of the plasmid; b. releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more programmable nicking enzyme recognizing the four target sites for the guide nucleic acid resulting in four nicks; d.
denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs; e. annealing the single strand DNA
overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d; f incubating the plasmid or the fragments resulting from step d with the restriction enzyme and thereby cleaving the plasmid or a fragment of the plasmid; and g. incubating the fragments of the plasmid with an exonuclease thereby digesting the fragments of the plasmid except the fragment resulting from step e.
1001241 In a further aspect, provided herein is a method for preparing a hairpin-ended DNA, wherein the method comprises: a. culturing a host cell comprising the plasmid of claim 24 under conditions resulting in amplification of the plasmid; b.
releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more programmable nicking enzyme recognizing the first, second, third, and fourth target sites for the guide nucleic acids resulting in four nicks; d. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;
e. annealing the single strand DNA overhangs intramolecularly and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step d; f. incubating the plasmid or the fragments resulting from step d with programmable nicking enzyme recognizing the fifth and sixth target sites for the guide nucleic acids resulting in the break in the double stranded DNA molecule; and g. incubating the fragments of the plasmid with an exonuclease thereby digesting the fragments of the plasmid except the fragment resulting from step e. In another embodiment, step f of the paragraph can be replaced with step f: incubating the plasmid or the fragments resulting from step d with one or more nicking endonuclease recognizing the two restriction sites resulting in the break in the double stranded DNA
molecule.
1001251 In certain embodiments, the DNA molecule that comprise an expression cassette flanked by inverted repeats (as described in Section 5.4) can be provided by culturing host cells comprising the DNA molecules or the plasmids and releasing the DNA
molecules or plasmid from the host cell as provided in the steps a and b in the preceding paragraphs.
Alternatively, such DNA molecules can be synthesized in a cell-free system or in a combination of cell-free and host cell-based systems. For example, chemical synthesis of DNA fragments and plasmids of various size and sequences is known and widely used in the art; fragments can be chemically synthesized and then ligated by any means known in the art, or recombined in a host cell. In other embodiments, the DNA molecules or plasmids can be provided by in vitro replication. Various methods can be used for in vitro replication, including amplification by polymerase chain reaction (PCR). PCR methods for replicating DNA fragments or plasmids of various sizes are well known and widely used in the art, for example, as described in Molecular Cloning: A Laboratory Manual, 4th Edition, by Michael Green and Joseph Sambrook, ISBN 978-1-936113-42-2 (2012), which is incorporated herein in its entirety by reference. In some embodiments, step a and b can be replaced by a step of providing DNA molecules by chemical synthesis or PCR. In other embodiments, step a, b, c, and d can be replaced by providing DNA molecules by chemical synthesis.

The order of the method steps are listed in the methods for illustrative purposes.
In certain embodiments, the method steps are performed in the order in which they appear in the claims. In some embodiments, the method steps can be performed in an order different from which they appear in the claims. Specifically, in some embodiments, the steps of the methods of making the hairpin-ended DNA molecules can be performed in the order as they appeared or as alphabetically listed in the claims, from a to e, or from a to g. Alternatively, the steps of the methods of making the hairpin-ended DNA molecules can be performed not in the order as they appear in the claims. In one embodiment, the step c (incubating the DNA
molecule with one or more nicking endonuclease recognizing the four restriction sites resulting in four nicks) can be performed before step b (releasing the plasmid from the host cell), when the host cells naturally express, are engineered to express, otherwise contain one or more nicking endonuclease. In another embodiment, step f (incubating the plasmid or the fragments resulting from step d with the restriction enzyme or incubating the plasmid or the fragments resulting from step d with one or more nicking endonuclease) can be performed before step d (denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs), or before step c (incubating the DNA molecule with one or more nicking endonuclease). Additionally, one or more steps can be combined into one step that perform all the actions of the separate step In certain embodiments, the step a (culturing a host cell) can be combined with step c (incubating the DNA molecule with one or more nicking endonuclease), when the host cells naturally express, are engineered to express, otherwise contain one or more nicking endonuclease. In other embodiments, step f (incubating the plasmid or the fragments resulting from step d with the restriction enzyme or incubating the plasmid or the fragments resulting from step d with one or more nicking endonuclease) can be combined with step c (incubating the DNA
molecule with one or more nicking endonuclease) by incubating with the nicking endonuclease or restriction enzyme recited in step f and c together.
Therefore, the disclosure provides that the steps can be performed in various combinations and permutations according to the state of the art.
1001271 Additional steps can be added to the methods provided herein, before all the method steps, after all the method steps, or in between any of the method steps. In one embodiment, the methods provided herein further include a step h. repairing the nicks with a ligase to form a circular DNA. In another embodiment, the step h of repairing the nicks with a ligase to form a circular DNA is performed after all the other method steps described herein.
1001281 As is further described further below in Sections 5.4.1 and 5.5, the hairpins formed at the end of the DNA molecules is determined by properties the overhang between the restriction sites for nicking endonucleases. Therefore, by designing the properties including the sequence and structural properties of the overhang between the restriction sites for nicking endonucleases according to Sections 5.4.1 and 5.5, the methods can be used to produce 1, 2 or more hairpinned ends. In one embodiment, the methods produce hairpin-ended DNA comprising 1 hairpin end. In another embodiment, the methods produce hairpin-ended DNA consisting of 1 hairpin end. In yet another embodiment, the methods produce hairpin-ended DNA comprising two hairpin ends. In a further embodiment, the methods produce hairpin-ended DNA consisting of two hairpin ends.
1001291 The methods provided herein can be used to produce DNA molecules comprising artificial sequences, natural DNA sequences, or sequences having both natural DNA
sequences and artificial sequences. In one embodiment, the methods produce hairpin-ended DNA molecules comprising artificial sequences. In another embodiment, the methods produce hairpin-ended DNA molecules comprising natural sequences. In yet another embodiment, the methods produce hairpin-ended DNA molecules comprising both natural sequences and artificial sequences. In certain embodiments, the methods produce hairpin-ended DNA molecules comprising viral inverted terminal repeat (ITR). In a further embodiment, the methods produce hairpin-ended DNA molecules comprising a viral genome In some embodiments, the viral genome is an engineered viral genome comprising one or more non-viral genes in the expression cassette. In certain embodiments, the viral genome is an engineered viral genome wherein one or more viral genes have been knocked out. In some specific embodiments, the viral genome is an engineered viral genome wherein the replication protein (Rep) gene, capsid (Cap) gene, or both Rep and Cap genes are knocked out. In other embodiments, the viral genome is parvovirus genome. In yet other embodiments, the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus.
1001301 The steps performed in the various methods provided herein are described in further details below. The embodiments of host cells and culturing of the host cells are described in Section 5.3.1; the embodiments for the step of releasing the DNA
molecules from the host cells are described in Section 5.3.2; the embodiments for the step of denaturing the DNA molecules are described in Section 5.3.3; the embodiments for the step of annealing are described in Section 5.3.5; the embodiments for the step of incubating the DNA
molecules with nicking endonucleases or restriction enzymes are described in Section 5.3.4;
the embodiments for the step of incubating with exonuclease are described in Section 5.3.6;
and the embodiments for the step of ligation are described in Section 53,7. As such, the disclosure provides methods comprising permutations and combinations of the various embodiments of the steps described herein.
5.3.1 Host Cells and Culturing of the Host cells [00131] The disclosure provides that various host cells can be cultured to amplify the DNA molecules. A host cell for use in the methods provided herein can be a eukaryotic host cell, a prokaryotic host cell, or any transformable organism that is capable of replicating or amplifying recombinant DNA molecules. In some embodiments, the host cell can be a microbial host cell. In further embodiments, the host cell can be a host microbial cell selected from, bacteria, yeast, fungus or any of a variety of other microorganism cells applicable to replicating or amplifying DNA molecules. A bacterial host cell can be that of any species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizohium etli, Bacillus ,subtilis, Corynehacteri urn glutamicum, Gluconohacter oxydans, Zymontonas mobilis, Lactococcus lactis, Lactobacillus plantarunt, Streptontyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. A
yeast or fungus host cell can be that of any species selected from Saccharomyces cerevisiae, Schizosaccharomyces pornbe, Khtyveromyces lactis, Khtyveromyces marxianus, Aspergilhts terreus, Aspergillus niger, Pichia pastor's, Rhizopu.s' arrhizus, Rhizobus oryzcte, and the like.
E. coil is a particularly useful host cell since it is a well characterized microbial cell and widely used for molecular cloning. Other particularly useful host cells include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host cells can be used to amplify the DNA molecules as known in the art.
[00132] Similarly, a eukaryotic host cell for use in the methods provided herein can be any eukaryotic cell that is capable of replicating or amplifying recombinant DNA
molecules, as known and used in the art. In some embodiments, a host cell for use in the methods provided herein can be a mammalian host cells. In further embodiments, a host cell can be a human or non-human mammalian host cell. In other embodiments, a host cell can be an insect host cell. Some widely used non-human mammalian host cells include CHO, mouse myeloma cell lines (e.g. NSO, SP2/0), rat myeloma cell line (e.g. YT32/0), and BHK.
Some widely used human host cells include HEK293 and its derivatives, HT-1080, PER.C6, and Huh-7. In certain embodiments, the host cell is selected from the group consisting of HeLa, NIH3T3, Jurkat, HEK293, COS, CHO, Saos, SF9, SF21, High 5, NSO, SP2/0, PC12, YB2/0, BHK, HT-1080, PER.C6, and Huh-7.
1001331 A host cell can be cultured as each host cell is known and cultured in the art. The culturing conditions and culture media for different host cells can be different as is known and practiced in the art. For example, bacterial or other microbial host cells can be cultured at 37 C, at an agitation speed of up to 300 rpm, and with or without forced aeration. Some insect host cells can be optimally cultured generally at 25 to 30 C, with no agitation at an agitation speed of up to 150 rpm, and with or without forced aeration. Some mammalian host cells can be optimally cultured at 37 C, with no agitation or at an agitation speed of up to 150 rpm, and with or without forced aeration. Additionally, conditions for culturing the various host cells can be determined by examining the growth curve of the host cells under various conditions, as is known and practiced in the art. Some widely used host cell culturing media and culturing conditions are described in Molecular Cloning- A
Laboratory Manual, 4th Edition, by Michael Green and Joseph Sambrook, ISBN 978-1-936113-42-2 (2012), which is incorporated herein in its entirety by reference.
5.3.2 Releasing the DNA molecules from Host Cells 1001341 DNA molecules can be released from the host cells by various ways as known and practiced in the art. For example, the DNA molecules can be released by breaking up the host cells physically, mechanically, enzymatically, chemically, or by a combination of physical, mechanical, enzymatic and chemical actions. In some embodiments, the DNA
molecules can be released from the host cells by subjecting the cells to a solution of cell lysis reagents. Cell lysis reagents include detergents, such as triton, SDS, Tween, NP-40, and/or CHAPS. In other embodiments, the DNA molecules can be released from the host cells by subjecting the host cells to difference in osmolarity, for example, subjecting the host cells to a hypotonic solution. In other embodiments, the DNA molecules can be released from the host cells by subjecting the host cells to a solution of high or low pH. In certain embodiments, the DNA molecules can be released from the host cells by subjecting the host cells to enzyme treatment, for example, treatment by lysozyme In some further embodiments, the DNA
molecules can be released from the host cells by subjecting the host cells to any combinations of detergent, osmolarity pressure, high or low pH, and/or enzymes (e.g.
lysozyme).
1001351 Alternatively, the DNA molecules can be released from the host cells by exerting physical force on the host cells. In one embodiment, the DNA molecules can be released from the host cells by directly applying force to the host cells, e.g. by using the Waring blender and the Polytron. Waring blender uses high-speed rotating blades to break up the cells and the Polytron draws tissue into a long shaft containing rotating blades. In another embodiment, the DNA molecules can be released from the host cells by applying shear stress or shear force to the host cells. Various homogenizers can be used to force the host cells through a narrow space, thereby shearing the cell membranes. In some embodiments, the DNA molecules can be released from the host cells by liquid-based homogenization. In one specific embodiment, the DNA molecules can be released from the host cells by use a Dounce homogenizer. In another specific embodiment, the DNA molecules can be released from the host cells by use a Potter-Elvehjem homogenizer. In yet another specific embodiment, the DNA molecules can be released from the host cells by use a French press.
Other physical forces to release the DNA molecules from host cells include manual grinding, e.g. with a mortar and pestle. In manual grinding, host cells are often frozen, e.g. in liquid nitrogen and then crushed using a mortar and pestle, during which process the tensile strength of the cellulose and other polysaccharides of the cell wall breaks up the host cells.
1001361 Additionally, the DNA molecules can be released from the host cells by subjecting the cells to freeze and thaw cycles. In some embodiments, a suspension of host cells is frozen and then thawed for a number of such freeze and thaw cycles.
In some embodiments, the DNA molecules can be released from the host cells by applying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 freeze and thaw cycles to the host cells.
1001371 The above described methods for releasing the DNA molecules from the host cells are not mutually exclusive. Therefore, the disclosure provides that the DNA
molecules can be released from the host cells by any combinations of DNA releasing methods provide in this Section 5.3.2.
5.3.3 Denaturing the DNA molecules 1001381 DNA molecules can be denatured by various ways as known and practiced in the art. The step of denaturing the DNA molecule can separate the DNA molecule from double strand DNA (dsDNA) into single strand DNA (ssDNA). In separating two DNA
strands, the temperature can be increased until the DNA unwinds and the hydrogen bonds that hold the two strands together weaken and finally break. The process of breaking double-stranded DNA into single strands is known as DNA denaturation, or DNA denaturing.
1001391 In some embodiments, the step of denaturing the DNA molecule can separate the two DNA strands of one or more segments of the dsDNA molecule, while keeping the other segment(s) of the DNA molecule as dsDNA. In some further embodiments, the step of denaturing the DNA molecules can separate the dsDNA into ssDNA at the segment between the first and second restriction sites for nicking endonuclease on the top and bottom strand of the DNA (e.g. DNA molecules described in Section 5.4), while keeping the other part of the DNA molecule as dsDNA, thereby creating an overhang between the first and second restriction sites. In certain embodiments, the step of denaturing the DNA
molecules can separate the dsDNA into ssDNA at the segment between the third and fourth restriction sites for nicking endonuclease on the top and bottom strand of the DNA (e.g. DNA
molecules described in Section 5.4), while keeping the other part of the DNA molecule as dsDNA, thereby creating an overhang between the third and fourth restriction sites.
In other embodiment, the step of denaturing the DNA molecules can separate the dsDNA
into ssDNA
at the segments between the first and second restriction sites and between the third and fourth restriction sites for nicking endonuclease on the top and bottom strand of the DNA (e.g. DNA
molecules described in Section 5.4), while keeping the other part of the DNA
molecule as dsDNA, thereby (1) breaking the DNA molecule into two daughter DNA molecules and (2) creating an overhang between the first and second restriction sites and an overhang between the third and fourth restriction sites. In one embodiments, the overhang between the first and second restriction sites for nicking endonuclease can be a top strand 5' overhang. In another embodiment, the overhang between the first and second restriction sites for nicking endonuclease can be a bottom strand 3' overhang. In yet another embodiment, the overhang between the third and fourth restriction sites for nicking endonuclease can be a top strand 3' overhang. In a further embodiment, the overhang between the third and fourth restriction sites for nicking endonuclease can be a bottom strand 5' overhang. In some embodiments, step of denaturing the DNA molecule can separate the DNA molecules in any combinations of the embodiments provided herein.
1001401 The overhang can vary in length depending on the distance between the restriction sites for nicking endonuclease. In one embodiment, the overhangs can be identical in length and/or sequences. In another embodiment, the overhangs can be different in length and/or sequences. In some embodiments, a top strand 5' overhang can be at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides in length. In other embodiments, a top strand 5' overhang can be about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, or more nucleotides in length.
In certain embodiments, a bottom strand 3' overhang can be at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides in length. In further embodiments, a bottom strand 3' overhang can be about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, or more nucleotides in length. In yet other embodiments, a top strand 3' overhang can be at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides in length. In other embodiments, a top strand 3' overhang can be about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, or more nucleotides in length.
In some embodiments, a bottom strand 5' overhang can be at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides in length. In other embodiments, a bottom strand 5' overhang can be about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, or more nucleotides in length.
1001411 As is known and practiced in the art, the DNA molecules can be denatured by heat, by changing the pH in the environment of the DNA molecules, by increasing the salt concentration, or by any combination of these and other known means The disclosure provides that the DNA molecules can be denatured in the methods by using a denaturing condition that selectively separates the dsDNA into ssDNA at the segments between the first and second restriction sites and/or between the third and fourth restriction sites on the top and bottom strand of the DNA, while keeping the other part of the DNA molecule as dsDNA.
Such selective separating of dsDNA to ssDNA can be performed by controlling the denaturing conditions and/or the time the DNA molecules are subjected to the denaturing conditions. In one embodiment, the DNA molecules are denatured at a temperature of at least 70 C, at least 71 C, at least 72 C, at least 73 C, at least 74 C, at least 75 C, at least 76 C, at least 77 C, at least 78 C, at least 79 C, at least 80 C, at least 81 C, at least 82 C, at least 83 C, at least 84 C, at least 85 C, at least 86 C, at least 87 C, at least 88 C, at least 89 C, at least 90 C, at least 91 C, at least 92 C, at least 93 C, at least 94 C, or at least 95 C. In another embodiment, the DNA molecules are denatured at a temperature of about 70 C, about 71 C, about 72 C, about 73 C, about 74 C, about 75 C, about 76 C, about 77 C, about 78 C, about 79 C, about 80 C, about 81 C, about 82 C, about 83 C, about 84 C, about 85 C, about 86 C, about 87 C, about 88 C, about 89 C, about 90 C, about 91 C, about 92 C, about 93 C, about 94 C, or about 95 C. In one specific embodiment, the DNA molecules are denatured at a temperature of about 90 C.
1001421 Other than denaturation by heat, sections or all the DNA molecules provided herein can undergo the denaturation process by addition of various chemical agents such as guanidine, formamide, sodium salicylate, dimethyl sulfoxide, propylene glycol, and urea.
These chemical denaturing agents lower the melting temperature by competing for hydrogen bond donors and acceptors with pre-existing nitrogenous base pairs and allow for isothermal denaturing. In some embodiments, chemical agents are able to induce denaturation at room temperature. In some specific embodiment, alkaline agents (e.g. NaOH) can be used to denature DNA by changing pH and removing hydrogen-bond contributing protons.
In other embodiments, chemically denaturing the DNA molecules provided herein can be a gentler procedure for DNA stability compared to denaturation induced by heat. In other embodiments, chemically denaturing and renaturing the DNA molecules (e.g.
changing the pH) provided herein can be a quicker than by heating. In some embodiments, the DNA of the disclosure can be replicated and nicked in bacteria and denatured simultaneously during the release (e.g. alkali lysis step) from bacteria.
1001431 In one embodiment, the DNA molecules are denatured at a pH of at least

10, at least 10.1, at least 10.2, at least 10.3, at least 10.4, at least 10.5, at least 10.6, at least 10.7, at least 10.8, at least 10.9, at least 11, at least 11.1, at least 112, at least

11.3, at least 11.4, at least 11.5, at least 11.6, at least 11.7, at least 11.8, at least 11.9, at least 12, at least 12.1, at least 12.2, at least 12.3, at least 12.4, at least 12.5, at least 13, at least 13.5, or at least 14. In another embodiment, the DNA molecules are denatured at a pH of about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12, about 12.1, about 12.2, about 12.3, about

12.4, about 12.5, about 13, about 13.5, or about 14. In yet another embodiment, the DNA
molecules are denatured at a salt concentration of at least 1M, at least 1.5M, at least 2M, at least 2.5M, at least 3M, at least 3.5M, or at least 4M of salt. In a further embodiment, the DNA molecules are denatured at a salt concentration of about 1M, about 1.5M, about 2M, about 2.5M, about 3M, about 3.5M, or about 4M of salt. In certain embodiments, the DNA molecule is subject to the denaturing condition for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 minutes. In other embodiments, the DNA molecule is subject to the denaturing condition for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 minutes. In some embodiments, the DNA molecules can be denatured by any combination of denaturing conditions and duration of denaturing as provided herein.
1001441 The denaturing conditions can be determined for the method step to selectively denaturing the segments between the first and second restriction sites and between the third and fourth restriction sites on the top and bottom strand of the DNA, while keeping the other part of the DNA molecule as dsDNA. Such selective denaturing conditions can be determined according to the properties of the DNA segments to be selectively denatured.
The stability of the DNA double helix correlates with the length of the DNA
segments and the percentage of G/C content. The disclosure provides that the selective denaturing conditions can be determined by the sequence of the DNA segments to be selectively denatured or the resulting sequence of the overhang. For example, the temperature for selective denaturing can be approximately determined as Tm = 2 C >< number of A-T pair +
4 C >< number of G-C pair for a DNA sequence to be selectively denatured.
Other more precise calculations of the Tm are also known and used in the art, for example, as described in Freier SM, eta., Proc Nall Acad Sci, 83, 9373-9377 (1986); Breslauer KJ, et al., Proc Nall Acad Sci, 83, 3746-3750 (1986); Panjkovich,A. and Melo,F. Bioinfortnatics 21:711-722 (2005); Panjkovich,A , et al. Nucleic Acids Res 33-W570-W572 (2005), all of which are herein incorporated in their entireties by reference.
1001451 The overhang can comprise various DNA sequences. In one embodiment, the overhang comprises inverted repeats. In another embodiment, the overhang comprises viral inverted repeats. In yet another embodiment, the overhang comprises or consists of any embodiments of sequences described in Sections 5.4.1, 5.4.2, 5.4.3, and 5.5.
In a further embodiment, the overhang comprises or consists of any one of the sequences as described in Sections 5.4.1 and 5.5.
5.3.4 Incubating the DNA Molecules With One or More Nicking Endonucleases or Restriction Enzymes 1001461 The disclosure provides one or more method steps for incubating the DNA
molecules with one or more nicking endonucleases or restriction enzymes as described in Sections 3 and 5.2. Without being bound by the theory, a nicking endonuclease recognizes the restriction sites for the nicking endonuclease in the DNA molecule and cuts only on one strand (e.g. hydrolyzes the phosphodiester bond of a single DNA strand) of the dsDNA at a site that is either within or outside the restriction sites for the nicking endonuclease, thereby creating a nick in the dsDNA. A restriction enzyme, on the other hand, recognizes the restriction sites for the restriction enzyme and cuts both strands of the dsDNA, thereby cleaving DNA molecules at or near the specific restriction sites.
1001471 In the various embodiments of compositions and methods provided herein, nicking endonucleases can be methylation-dependent, methylation-sensitive, or methylation-insensitive. Various nicking endonucleases known and practiced in the art are provided herein. In some embodiments, the nicking endonucleases for the compositions and methods provided herein can be naturally occurring nicking endonucleases that are not methylcytosine dependent, including Nb.Bsml, Nb.BbvCI, Nb.BsrDI, Nb.Btsl, Nt.BbvCI, Nt.Alwl, Nt. CviPII, Nt. BsmAI, Nt. Alwl and Nt.BstNBI. Nicking endonucleases for the compositions and methods provided herein can also be engineered from Type IIs restriction enzymes (e.g., Alwl, Bpul0I, BbvCI, Bsal, BsmBI, BsmAI, Bsml, Bsp0J, Mlyl, Mval2691 and Sapl, etc.) and methods of making nicking endonucleases can be found in references for example in, US 7,081,358; US 7,011,966; US 7,943,303; US 7,820,424, W0201804514, all of which are herein incorporated in their entirety by reference.
1001481 Alternatively, a programmable nicking enzyme can be used for the compositions and methods provided herein instead of nicking endonucleases. Such programmable nicking enzyme include, e.g., Cas9 or a functional equivalent thereof (such as Pyrococcus furiosus Argonaute (PfAgo) or Cpfl). Cas9 contains two catalytic domains, RuvC and HNH.

Inactivating one of those domains will generate a programmable nicking enzyme that can replace a nicking endonuclease for the methods and compositions provided herein. In Cas9, the RuvC domain can be inactivated by an amino acid substitution at position D10 (e.g., D10A) and the HNH domain can be inactivated by an amino acid substitution at position H840 (e.g., H840A), or at a position corresponding to those amino acids in other Cas9 equivalent proteins. Such programmable nicking enzyme can also be Argonaute or Type II
CRISPR/Cas endonucleases that comprise two components: a nicking enzyme (e.g., a DlOA
Cas9 nicking enzyme or variant or ortholog thereof) that cleaves the target DNA and a guide nucleic acid e.g., a guide DNA or RNA (gDNA or gRNA) that targets or programs the nicking enzyme to a specific site in the target DNA (see, e.g., Hsu, et al., Nature Biotechnology 2013 31: 827-832, which is herein incorporated in its entirety by reference).
A programmable nicking enzyme can also be made by fusing a site specific DNA
binding domain (targeting domain) such as the DNA binding domain of a DNA binding protein (e.g., a restriction endonuclease, a transcription factor, a zinc-finger or another domain in that binds to DNA at non-random positions) with a nicking endonuclease so that it acts on a specific, non-random site. As is clear from the foregoing, the programmable cleavage by a programmable nicking enzyme results from targeting domain within or fused to the nicking enzyme or from guide molecules (gDNA or gRNA) that direct the nicking enzyme to a specific, non-random site, which site can be programmed by changing the targeting domain or the guide molecule. Such programmable nicking enzymes can be found in references for example, US 7,081,358 and W02010021692A, which are herein incorporated in their entireties by reference.
1001491 Suitable guide nucleic acid (e.g. gDNA or gRNA) sequences and suitable target sites for the guide nucleic acid have been known and widely utilized in the art. The guide nucleic acid (e.g. gDNA or gRNA) is a specific nucleic acid (e.g. gDNA or gRNA) sequence that recognizes the target DNA region of interest and directs the programmable nicking enzyme (e.g. Cas nuclease) there for editing. The guide nucleic acid (e.g.
gDNA or gRNA) is often made up of two parts: targeting nucleic acid, a 15-20 nucleotide sequence complementary to the target DNA, and a scaffold nucleic acid, which serves as a binding scaffold for the programmable nicking enzyme (e.g. Cas nuclease). The suitable target sites for the guide nucleic acid must have two components the complementary sequence to the targeting nucleic acid in the programmable nicking enzyme and an adjacent Protospacer Adjacent Motif (PAM) The PAM serves as a binding signal for the programmable nicking enzyme (e.g. Cas nuclease). Various PAMs have been known, characterized, and utilized in the art, for example as discussed in Daniel Gleditzsch et al., RNA Biol.
16(4): 504-517 (April 2019); Ryan T. Leenay et al., Mol Cell. 62(1): 137-147 (Apr 7, 2016), both of which are herein incorporated in their entirety by reference. Exemplary gRNA and gDNA
sequences targeting the primary stem sequence of AAV2 ITRs include such listed in Table 1.
Table 1: Exemplary Nicking Endonuclease and Their Corresponding Restriction Sites SEQ ID NO:176 AGCGAGCGAGCGCGCAGAGAGGG
AAV2 wt gRNA for Nicking Cas9 SEQ ID NO:177 GCTCGCTCGCTCGGTG
AAV2 wt gDNA for PfAgo 1001501 Various nicking endonucleases known and used in the art can be used in the methods provided herein. An exemplary list of nicking endonuclease provided as embodiments for the nicking endonuclease for use in the methods and the corresponding restriction sites for some of the nicking endonuclease are described in The Restriction Enzyme Database (known in the art as REBASE), which is available at www.rebase.neb.com/cgi-bin/azlist?nick and incorporated herein in its entirety by reference.
In one embodiments, the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site are all for target sequences for the same nicking endonuclease. In another embodiment, the first, second, third, and fourth restriction sites for nicking endonucleases are target sequences for two different nicking endonucleases, including all possible combinations of arranging the four sites for two different nicking endonuclease target sequences (e.g. the first restriction site for the first nicking endonuclease and the rest for the second nicking endonuclease, the first and second restriction sites for the first nicking endonuclease and the rest for the second nicking endonuclease etc.). In yet another embodiment, the first, second, third, and fourth restriction sites for nicking endonucleases are target sequences for three different nicking endonucleases, including all possible combinations of arranging the four sites for three different endonuclease target sequences. In a further embodiment, the first, second, third, and fourth restriction sites for nicking endonucleases are target sequences for four different nicking endonucleases.
In some embodiments, the nicking endonuclease can be any one selected from those listed in Table 2.
Table 2: Exemplary Nicking Endonuclease and Their Corresponding Restriction Sites:
Nicking Corresponding Restriction Sites for the Nicking Endonuclease and Endonuclease Position of Nick Relative to the Restriction Sites (Note: 1/none means the nick is 1 nucleotide 3' from the restriction sites on the top strand).
Nt. Bsm AI GTCTC (1/none) Nt. BtsCI GGATG (2/none) N. ALwl GGATC (4/none) N. BstNBI GAGTC (4/none) N. BspD6I GAGTC (4/none) Nb. Mval269I GAATGC (none/-1) Nb. BsrDI GCAATG (none/0) Nb. BtsI GCAGTG (none/0) Nt. BtsI GCAGTG (2/none) Nt. BsaI GGTCTC (1/none) Nt. BpulOI CCTNAGC (-5/none) Nb .Bpu 1 OI CCTNAGC (none/-2) Nt. BsmBI CGTCTC (1/none) Nb. BbvCI CCTCAGC (none/-2) Nt. BbvCI CCTCAGC (-5/none) Nt. BspQI GCTCTTC (1/none) 1001511 The conditions for the various nicking endonuclease to cut one strand of the dsDNA are known for the various nicking endonucleases provided herein, including the temperatures, the salt concentration, the pH, the buffering reagent, the presence or absence of certain detergent, and the duration of incubation to achieve the desired percentage of nicked DNA molecules. These conditions are readily available from the websites or catalogs of various vendors of the nicking endonucleases, e.g. New England BioLabs. The disclosure provides that the step of incubating the DNA molecule with one or more nicking endonuclease is performed according to the incubation conditions as known and practiced in the art.
1001521 Various restriction enzymes known and used in the art can be used in the methods provided herein. An exemplary list of restriction enzymes provided as embodiments for the restriction enzymes for use in the methods and the corresponding restriction sites for the restriction enzymes are described in the catalog of New England Biolabs, which is available at neb.com/products/restriction-endonucleases and incorporated herein in its entirety by reference. The conditions for the various restriction enzymes to cleave the dsDNA are known for the various restriction enzymes provided herein, including the temperatures, the salt concentration, the pH, the buffering reagent, the presence or absence of certain detergent, and the duration of incubation to achieve the desired percentage of nicked DNA
molecules.
These conditions are readily available from the websites or catalogs of various vendors of the restriction enzymes, e g New England BioLabs The disclosure provides that the step of incubating the DNA molecule with the restriction enzymes is performed according to the incubation conditions as known and practiced in the art.
5.3.5 Annealing 1001531 The step of annealing in the methods provided herein is performed to selectively anneal the ssDNA overhang intramolecularly and thereby creating a hairpinned inverted repeat on one end of the DNA fragment (e.g. from Sections 5.4 and 5.5) resulted from the step of denaturing as described above (Section 5.3.3). In certain embodiments, the step of annealing in the methods provided herein is performed to selectively anneal the ssDNA
overhangs intramolecularly and thereby creating hairpinned inverted repeats on two ends the DNA fragment (e.g. from Sections 5.4 and 5.5) resulted from the step of denaturing as described above (Section 5.3.3). Without being bound or otherwise limited by the theory, such selective intramolecular annealing of the ssDNA overhangs is achieved because the intramolecular complementary sequences within the ssDNA overhangs make the intramolecular annealing of the ssDNA overhangs thermodynamically and/or kinetically favored over the intermolecular annealing of the ssDNA overhangs.
1001541 Without being bound or otherwise limited by the theory, it is recognized that certain lengths and/or the sequences of the overhang can make the intramolecular annealing of the ssDNA overhangs thermodynamically and/or kinetically favored over the intermolecular annealing of the ssDNA overhangs. For example, a linear interaction plot showing the intramolecular forces within the overhang and intermolecular forces between the strands as well as the resulting structure is depicted in FIG. 2A-C. The thermodynamics and the kinetics of the annealing of the ssDNA overhang is determined by the enthalpy (AH) and the entropy (AS), among other factors. The inventors recognize that, as the loss of movement freedom from a free ssDNA overhang to an intramolecularly annealed overhang is less than the loss of movement freedom from free ssDNA overhang to intermolecularly annealed overhang, the entropy loss in an intramolecular annealing is less than the entropy loss in an intramolecular annealing. On the other hand, as the number of complementary nucleotide pairs in an intramolecularly annealed overhang is less than number of complementary nucleotide pairs in an intermolecularly annealed overhang (hence less Watson-Crick and Hoogsteen-type hydrogen bonding), the enthalpy gain in an intramolecular annealing may be less than the enthalpy gain in an intramolecular annealing. The disclosure provides that the ssDNA overhang can be designed to have certain lengths, numbers of complementary nucleotide pairs, and percentage of G-C and A-T pairs, such that the free energy gain (AG=
AH¨TAS) of intramolecular annealing of the overhang is bigger over that of intermolecular annealing, thereby making the intramolecular annealing thermodynamically favored over the intermolecular annealing. The inventors further recognize that, as the nucleotides within the ssDNA overhang have a higher probability of contacting each other than contacting the nucleotides of another ssDNA overhang in molecular motion, the kinetics of intramolecular annealing of the ssDNA overhang can be higher than that of intermolecular annealing. The disclosure provides that even if the intramolecular annealing is thermodynamically disfavored over the intermolecular annealing, the superior kinetics of intramolecular annealing of the ssDNA overhang can result in the formation of intramolecularly annealed overhang over intermolecularly annealed overhang.
1001551 The annealing step can be performed at various temperatures to favor the intramolecular annealing over intermolecular annealing. In one embodiment, the ssDNA
overhang is annealed at a temperature of at least 15 C, at least 16 C, at least 17 C, at least 18 C, at least 19 C, at least 20 C, at least 21 C, at least 22 C, at least 23 C, at least 24 C, at least 25 C, at least 26 C, at least 27 C, at least 28 C, at least 29 C, at least 30 C, at least 31 C, at least 32 C, at least 33 C, at least 34 C, at least 35 C, at least 36 C, at least 37 C, at least 38 C, at least 39 C, at least 40 C, at least 41 C, at least 42 C, at least 43 C, at least 44 C, at least 45 C, at least 46 C, at least 47 C, at least 48 C, at least 49 C, at least 50 C, at least 51 C, at least 52 C, at least 53 C, at least 54 C, at least 55 C, at least 56 C, at least 57 C, at least 58 C, at least 59 C, or at least 60 C. In another embodiment, the ssDNA overhang is annealed at a temperature of about 15 C, about 16 C, about 17 C, about 18 C, about 19 C, about 20 C, about 21 C, about 22 C, about 23 C, about 24 C, about 25 C, about 26 C, about 27 C, about 28 C, about 29 C, about 30 C, about 31 C, about 32 C, about 33 C, about 34 C, about 35 C, about 36 C, about 37 C, about 38 C, about 39 C, about 40 C, about 41 C, about 42 C, about 43 C, about 44 C, about 45 C, about 46 C, about 47 C, about 48 C, about 49 C, about 50 C, about 51 C, about 52 C, about 53 C, about 54 C, about 55 C, about 56 C, about 57 C, about 58 C, about 59 C, or about 60 C. In one specific embodiment, the ssDNA overhang is annealed at a temperature of at least 25 C. In another specific embodiment, the ssDNA overhang is annealed at a temperature of about 25 C. In yet another specific embodiment, the ssDNA
overhang is annealed at room temperature.
1001561 Additionally, the annealing step can be performed for various durations of time to favor the intramolecular annealing over intermolecular annealing. In certain embodiments, the ssDNA overhang is annealed for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 minutes. In other embodiments, the ssDNA
overhang is annealed for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 minutes. In one specific embodiment, the ssDNA
overhang is annealed for at least 20 minutes. In another specific embodiment, the ssDNA
overhang is annealed for about 20 minutes.
1001571 In some embodiments, annealing can be accomplished by lowering the temperature below the calculated melting temperatures of the sense and antisense sequence pairs. The melting temperature is dependent upon the specific nucleotide base content and the characteristics of the solution being used, e.g., the salt concentration.
Melting temperatures for any given sequence and solution combination are readily calculated as known and practiced in the art.
1001581 In some embodiments, annealing can be accomplished isothermally by reducing the amount of denaturing chemical agents to allow an interaction between the sense and antisense sequence pairs. The minimum concentration of denaturing chemical agents required to denature the DNA sequence can dependent upon the specific nucleotide base content and the characteristics of the solution being used, e.g., temperature or the salt concentration. The concentration of chemical denaturing agents that do not lead to denaturing for any given sequence and solution combination are readily identified as known and practiced in the art. The concentration of chemical denaturing agents can also be readily modified as known and practiced in the art. For example, the amount of urea can be lowered by dialysis or tangential flow filtration or the pH can be changed by the addition of acids or bases.
1001591 The annealing temperature and the annealing duration for intramolecular annealing correlate with the lengths of the ssDNA overhang, the number of complementary nucleotide pairs, and percentage of G-C and A-T pairs, and the sequence of the ssDNA
overhang (the arrangement of the complementary nucleotide pairs). In certain embodiments, an ssDNA overhang provided for the methods provided herein comprises any number of nucleotides in length as described in Section 5.3.3. In certain embodiments, a ssDNA
overhang provided for the methods provided herein comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 intramolecularly complementary nucleotide pairs. In some embodiments, a ssDNA overhang provided for the methods provided herein comprises about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about intramolecularly complementary nucleotide pairs. In some embodiments, a ssDNA
overhang provided for the methods provided herein comprises at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% G-C pairs among intramolecularly complementary nucleotide pairs. In certain embodiments, a ssDNA overhang provided for the methods provided herein comprises about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% G-C pairs among intramolecularly complementary nucleotide pairs.
[00160] Additionally, the inventors recognize that the concentration of the DNA
molecules, which correlates with the concentration of the overhangs, can affect the equilibrium and kinetics of the intramolecular annealing and the intermolecular annealing of the overhangs. Without being bound or otherwise limited by the theory, when the concentration of the overhang is too high, the probability of the intermolecular contact among the overhangs increases and the kinetic advantage of the intramolecular contact over intermolecular contact seen at lower concentration as discussed above is then diminished.
[00161] As discussed above, in some embodiments, intramolecular interactions can occur at a faster rate while intermolecular interactions occur at a slower rate. In some embodiments, base pair interactions involving three or more molecules (e.g.
three different strands) occur at the slowest rate. In some embodiments, the kinetic rate of intramolecular interactions versus intermolecular interactions is governed by the concentration of each molecule. In some embodiments, the intramolecular interactions are kinetically faster or intramolecular forces are larger when the concentration of DNA strands is lower.
[00162] Viewed individually, the absolute free energy of forming each complementary domain of IRs or ITRs, may be different, leading to regions of the IR or ITR
that may locally fold earlier as the strand transitions from a denatured to annealed state. The presence of locally folded domains (e.g. a central hairpin or branched hairpin like in A
AV2 ITRs as described in elsewhere in this Section (Section 5.4.1) and Section 5.5) can reduce the amount of bases available for pairing with other strands and thus can reduce the likelihood of intermolecular annealing or hybridization and shift the equilibrium from intermolecular annealing to intramolecular annealing or ITR formation.
[00163] Accordingly, the disclosure provides that the annealing step can be performed at various concentrations to favor the intramolecular annealing over intermolecular annealing.
In some embodiments, the ssDNA overhang is annealed at a concentration of no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, no more than 100, no more than 110, no more than 120, no more than 130, no more than 140, no more than 150, no more than 160, no more than 170, no more than 180, no more than 190, no more than 200, no more than 210, no more than 220, no more than 230, no more than 240, no more than 250, no more than 260, no more than 270, no more than 280, no more than 290, no more than 300, no more than 325, no more than 350, no more than 375, no more than 400, no more than 425, no more than 450, no more than 475, no more than 500, no more than 550, no more than 600, no more than 650, no more than 700, no more than 750, no more than 800, no more than 850, no more than 900, no more than 950, no more than 1000 ng/p.1 for the DNA
molecules. In certain embodiments, the ssDNA overhang is annealed at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000 ng4t1 for the DNA molecules.
1001641 Similarly, the disclosure provides that the annealing step can be performed at various molar concentrations to favor the intramolecular annealing over intermolecular annealing. In some embodiments, the ssDNA overhang is annealed at a concentration of no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, no more than 100, no more than 110, no more than 120, no more than 130, no more than 140, no more than 150, no more than 160, no more than 170, no more than 180, no more than 190, no more than 200, no more than 210, no more than 220, no more than 230, no more than 240, no more than 250, no more than 260, no more than 270, no more than 280, no more than 290, no more than 300, no more than 325, no more than 350, no more than 375, no more than 400, no more than 425, no more than 450, no more than 475, no more than 500, no more than 550, no more than 600, no more than 650, no more than 700, no more than 750, no more than 800, no more than 850, no more than 900, no more than 950, no more than 1000 nM for the DNA molecules. In certain embodiments, the ssDNA overhang is annealed at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000 nM for the DNA molecules. In some further embodiments, the ssDNA overhang is annealed at a concentration of no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20 ti.M. In yet other embodiments, the ssDNA overhang is annealed at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 tiM. In one specific embodiment, the ssDNA overhang is annealed at a concentration of about 10 nM for the DNA molecules. In another specific embodiment, the ssDNA overhang is annealed at a concentration of about 20 nM for the DNA
molecules. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 30 nM for the DNA molecules. In a further specific embodiment, the ssDNA
overhang is annealed at a concentration of about 40 nM for the DNA molecules. In still another specific embodiment, the ssDNA overhang is annealed at a concentration of about 50 nM
for the DNA molecules In another specific embodiment, the ssDNA overhang is annealed at a concentration of about 60 nM for the DNA molecules. In one specific embodiment, the ssDNA overhang is annealed at a concentration of about 10 ng/til for the DNA
molecules. In another specific embodiment, the ssDNA overhang is annealed at a concentration of about 20 ng/ttl for the DNA molecules. In yet another specific embodiment, the ssDNA
overhang is annealed at a concentration of about 30 ng/ .1 for the DNA molecules. In a further specific embodiment, the ssDNA overhang is annealed at a concentration of about 40 ng/til for the DNA molecules. In one specific embodiment, the ssDNA overhang is annealed at a concentration of about 50 ng/til for the DNA molecules. In another specific embodiment, the ssDNA overhang is annealed at a concentration of about 60 ng/til for the DNA
molecules. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 70 ng/til for the DNA molecules. In one specific embodiment, the ssDNA
overhang is annealed at a concentration of about 80 ng/ .1 for the DNA molecules. In another specific embodiment, the ssDNA overhang is annealed at a concentration of about 90 ng/til for the DNA molecules. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 100 ng/til for the DNA molecules.

1001651 In some embodiments, an ssDNA overhang provided for the methods provided herein comprises any sequences listed in Table 3.
Table 3: Sequences of ssDNA overhang and the corresponding structure after annealing.
ssDNA overhang sequences Structures after annealing SEQ ID NO:3 FIG. 3A
SEQ ID NO:4 FIG. 3A
SEQ ID NO:5 FIG. 3A
SEQ ID NO:7 FIG. 3B
SEQ ID NO:8 FIG. 3B
SEQ ID NO:9 FIG. 3B
SEQ ID NO:10 FIG. 3B
SEQ ID NO:33 FIG. 3C
SEQ ID NO:34 FIG. 3C
SEQ ID NO:35 FIG. 3C
SEQ ID NO:27 FIG. 5 SEQ ID NO:29 FIG. 4 SEQ ID NO:28 FIG. 4 1-1BOV (nucleotides 129-237 on wt genome) FIG. 1 B19 (nucleotides 139-227 on wt genome) FIG. 1 1001661 In some embodiments, the structure of the DNA molecules provided herein is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing (e.g.
denaturing as described in Section 5.3.3 and re-annealing as described in this Section (Section 53.5)).
DNA structures can be described by an ensemble of structures at or around the energy minimum.
In certain embodiments, the ensemble DNA structure is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing. In one embodiment, the folded hairpin structure formed from the ITR
or IR provided herein is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing. In another embodiment, the ensemble structure of the folded hairpin is the same after 2, 3, 4, 5, or 20 cycles of denaturing/renaturing.
5.3.6 Incubating with Exonuclease 1001671 The disclosure provides a step of incubating with an exonuclease as described in Section 3. Exonucleases cleaves nucleotides from the end (exo) of a DNA
molecules.
Exonucleases can cleave nucleotides along the 5' to 3' direction, along the 3' to 5' direction, or along both directions. In certain embodiments, an exonuclease for use in the methods provided herein cleaves nucleotides with no sequence specificity In some embodiments, an exonuclease for use in the methods provided herein digests the DNA fragments comprising ends created by one or more nicking endonuclease recognizing and cutting the fifth and sixth restriction sites or by restriction enzyme cleaving the plasmid or a fragment of the plasmid, as provided in Section 3.
1001681 Various exonucleases known and used in the art can be used in the methods provided herein. An exemplary list of exonucleases provided as embodiments for the restriction enzymes for use in the methods are described in the catalog of New England Biolabs, which is available at neb.com/products/dna-modifying-enzymes-and-cloning-technologies/nucleases and incorporated herein in its entirety by reference.
The conditions for the various exonucleases to digest the DNA molecules are known for the various exonucleases provided herein, including the temperatures, the salt concentration, the pH, the buffering reagent, the presence or absence of certain detergent, and the duration of incubation to achieve the desired percentage of digestion. These conditions are readily available from the websites or catalogs of various vendors of the restriction enzymes, e.g.
New England BioLabs. The disclosure provides that the step of incubating the DNA molecule with the restriction enzymes is performed according to the incubation conditions as known and practiced in the art.
1001691 The step of incubating exonucleases selectively digests the DNA
molecules with one or more ends, while leaving the hairpin-ended DNA molecules intact. As is clear from the description of Sections 5.3.5 and 5.5, the hairpin-ended DNA molecules comprise 0, 1, 2, or more nicks. In some embodiments, an exonuclease for use in the methods provided herein can be an exonuclease that selectively digests DNA molecules with one or more ends, while leaving intact the circular ssDNA/dsDNA molecules or DNA molecules comprising one or more nicks but no ends. In one embodiment, an exonuclease for use in the methods provided herein can be Exonuclease V (RecBCD). In one embodiment, an exonuclease for use in the methods provided herein can be Exonuclease VIII or truncated Exonuclease VIII.

Exonuclease V (RecBCD), Exonuclease VIII, and truncated Exonuclease VIII
comprise the selectivity described in this paragraph. Other suitable exonucleases are also known, used in the art, and provided herein, for example, as described on the websites or in the catalogs of various vendors of exonucleases including New England BioLabs.
1001701 In some embodiments, after exonuclease treatment, the DNA molecules of the present disclosure are substantially free of any prokaryotic backbone sequences. In some embodiments, the backbone refers to the plasmid sequence that is not part of the sequence encompassing the expression cassette in between the two ITRs. In some embodiments, the backbone refers to the vector sequence that is not part of the sequence encompassing the expression cassette in between the two ITRs. In some embodiments, the isolated DNA
molecules of the disclosure are 100% free, 99% free, 98% free, 97% free, 96%
free, 95%
free, 94% free, 93% free, 92% free, 91% free, or 90% free of prokaryotic backbone sequence of the parental plasmid.
5.3.7 Repairing the Nicks with a Ligase 1001711 The disclosure provides a step of repairing the nicks with a ligase as described in Section 3. DNA ligases catalyze the joining of two ends of DNA molecules by forming one or more new covalent bonds. For example, commonly used T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in DNA. The formation of new covalent bonds that are catalyzed by ligase to joint two DNA
molecules is referred to as "ligation." In certain embodiments, a DNA ligase for use in the methods provided herein ligates nucleotides with no sequence specificity In some embodiments, a DNA ligase for use in the methods provided herein ligates the two ends at one nick of the DNA molecule described in Section 5.5, thereby repairing said one nick. In some embodiments, a DNA ligase for use in the methods provided herein ligates each pair of two ends at the two nicks of the DNA molecule described in Section 5.5, thereby repairing the two nicks. In some embodiments, a DNA ligase for use in the methods provided herein ligates each pair of two ends at all nicks of the DNA molecule described in Section 5.5, thereby repairing all nicks of the DNA molecule. When the DNA molecule described in Section 5.5 forms a circular DNA after all nicks of the DNA molecule described in Section 5.5 have been repaired. As described in Section 5.5, in some embodiments, the DNA
molecule described in Section 5.5 consists of two nicks. In certain embodiments, the DNA
molecule described in Section 5.5 comprises two nicks. In other embodiments, the DNA
molecule described in Section 5.5 consists of one nick. In yet other embodiments, the DNA
molecule described in Section 5.5 comprises one nick.
1001721 The disclosure provides that the step of repairing the nicks with a ligase is performed according to the incubation conditions as known and practiced in the art.
5.4 DNA Molecules Used in the Methods 1001731 The DNA molecule provided herein can be a DNA molecule in its native environment or an isolated DNA molecule. In certain embodiments, the DNA
molecule is a DNA molecule in its native environment. In some embodiments, the DNA molecule is an isolated DNA molecule. In one embodiment, the isolated DNA molecule can be a DNA

molecule of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% purity. In another embodiment, the isolated DNA molecule can be a DNA
molecule of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%
purity. Other embodiments of the isolated DNA molecules provided herein in terms of purities are further described in Section 5.4.8, which can be combined in any suitable combination with the embodiments provided in this paragraph.
1001741 As the DNA molecules can be fully engineered (e.g.
synthetically produced or recombinantly produced), the DNA molecules provided herein including those of Sections 3 and this Section 5.4 can lack certain sequences or features as further described in Section 5.4.5.

5.4.1 Inverted Repeats 1001751 The ITRs or IRs provided in Sections 3 and this Section (Section 5.4.1) can form the hairpinned ITRs in the hairpin-ended DNA molecules provided in Section 5.5, for example upon performing the method steps described in Sections 3, 5.3.3, 5.3.4, and 5.3.5.
Accordingly, in some embodiments, the ITRs or IRs provided in Sections 3 and this Section (Section 5.4.1) can comprise any embodiments of the IRs or ITRs provided in Sections 3 and Section 5.5 and additional embodiments provided in this Section (Section 5.4.1), in any combination.
1001761 "Inverted repeat" or "IR" refers to a single stranded nucleic acid sequence that comprises a palindromic sequence region. This palindromic region comprises a sequence of nucleotides as well as its reverse complement, i.e., "palindromic sequence" as further described below, on the same strand as further described below. In a denatured state, meaning in conditions in which the hydrophobic stacking attractions between the bases are broken, the IR nucleic acid sequence is present in a random coil state (e.g.
at high temperature, presence of chemical agents, high pH, etc.). As conditions become more physiological, said IR can fold into a secondary structure whose outermost regions are non-covalently held together by base pairing. In some embodiments, an IR can be an ITR. In certain embodiments, an IR comprise an ITR.
1001771 "Inverted terminal repeat" "terminal repeat," "TR," or "ITR" refers to an inverted repeat region that is at or proximal to a terminal of a single strand DNA
molecule or an inverted repeat that is at or in the single strand overhang of a dsDNA
molecule. An ITR can fold onto itself as a result of the palindromic sequence in the ITR. In one embodiment, an ITR is at or proximal to one end of an ssDNA. In another embodiment, an ITR is at or proximal to one end of a dsDNA. In yet another embodiment, two ITRs are each at or proximal to the two respective ends of an ssDNA. In a further embodiment, two ITRs are each at or proximal to the two respective ends of a dsDNA. In some embodiments, the non-ITR part of the ssDNA or dsDNA is heterologous to the ITR. In certain embodiments, the non-ITR part of the ssDNA or dsDNA is homologous to the ITR. In a denatured state, meaning in conditions in which the hydrophobic stacking attractions between the bases are broken, the ITR comprising nucleic acid sequence is present in a random coil state (e.g. at high temperature, presence of chemical agents, high pH, etc.). In some embodiments, as conditions become more suitable for annealing as described in Section 5.3.5, the ITR can fold on itself into a structure that is non-covalently held together by base pairing while the heterologous non-ITR part of the dsDNA remain intact or the heterologous non-ITR part of the ssDNA molecule can hybridize with a second ssDNA molecule comprising the reverse complement sequence of the heterologous DNA molecule. The resulting complex of two hybridized DNA strands encompass three distinct regions, a first folded single stranded ITR
covalently linked to a double stranded DNA region that is in turn covalently linked to a second folded single stranded ITR. In certain embodiments, the ITR sequence can start at one of the restriction site for nicking endonuclease described in Sections 3, 5.3.4, and 5.4.2 and end at the last base before the dsDNA. In one embodiment, as opposed to a linear double stranded DNA molecule, the ITR present at the 5' and 3' termini of the top and bottom strand at either end of the DNA molecule can fold in and face each other (e.g. 3' to 5', 5' to 3' or vice versa) and therefore do not expose a free 5' or 3' terminus at either end of the nucleic acid duplex. When the ITR folds on itself, the dsDNA in the folded ITR can be immediately next to the dsDNA of the non-ITR part of the DNA molecule, creating a nick flanked by dsDNA
in some embodiments, or the dsDNA in the folded ITR can be one or more nucleotide apart from the dsDNA of the non-ITR part of the DNA molecule, creating a "ssDNA gap"
flanked by dsDNA in other embodiments. The two ITRs that flank the non-ITR DNA
sequence are referred to an "ITR pair-. In some embodiments, when the ITR assumes its folded state, it is resistant to exonuclease digestion (e.g. exonuclease V), e.g. for over an hour at 37 C.
1001781 The boundary between the terminal base of the ITR folded into its secondary structure and the terminal base of the DNA hybridized duplex can further be stabilized by stacking interactions (e.g. coaxial stacking) between base pairs flanking the nick or ssDNA
gap and these interactions are sequence-dependent. In the case of a structure resembling a nick, an equilibrium between two conformations can exist wherein, the first conformation is very close to that of the intact double helix where stacking between the base pairs flanking the nick is conserved while the other conformation corresponds to complete loss of stacking at the nick site thus inducing a kink in DNA. Nicked molecules are known to move somewhat slower during polyacrylamide and agarose gel electrophoresis than intact molecules of the same size. In some cases, this retardation is enhanced at higher temperatures. It is thought that the fast equilibration between stacked/straight and unstacked/bent conformations of the nick directly affects the mobility of DNA
molecule during gel electrophoreses, leading to differential retardation characteristic to a DNA
molecule carrying the nick.
1001791 Without being bound by theory, it is thought that cellular proteins can recognize parallel 5' and 3' termini as double strand breaks and can engage as well as process these, which can adversely affect the fate of the DNA in a cell. Hence, the ITR can prevent premature, unwanted degradation of the expression cassette with ITRs at one or both of its two ends as provided in Sections 3 and 5.5 and this Section (Section 5.4.1).
1001801 By placing a first and a second restriction site for nicking endonucleases on opposite strands and in proximity of the inverted repeats and subsequent separation of the top from the bottom strand of the inverted repeat, the resulting overhang can fold back on itself and form a double stranded end that contains at least one restriction site for the nicking endonuclease. In some embodiments, the folded ITR resembles the secondary structure conformation of viral ITRs. In one embodiment, the ITR is located on both the 5' and 3' terminus of the bottom strand (e.g. a left ITR and right ITR). In another embodiment, the ITR is located on both the 5' and 3' terminus of the top strand. In yet another embodiment, one ITR is located at the 5' terminus of the top strand, and the other ITR is located at the opposite end of the bottom strand (e.g. the left ITR at the 5' terminus on the top strand and the right ITR at the 5' terminus of the bottom) In yet another embodiment, one ITR is located at the 3' terminus of the top strand, and the other ITR is located at the 3' terminus of the bottom strand.
1001811 In some aspects, the disclosure provides a DNA molecule comprising palindromic sequences. "Palindromic sequences" or "palindromes" are self-complimentary DNA

sequences that can fold back to form a stretch of dsDNA in the self-complimentary region under a condition that favors intramolecular annealing. In some embodiments, a palindromic sequence comprises a contiguous stretch of polynucleotides that is identical when read forwards as when read backwards on the complementary strand. In one embodiment, a palindromic sequence comprises a stretch of polynucleotides that is identical when read forwards as when read backwards on the complementary strand, wherein such stretch is interrupted by one or more stretches of non-palindromic polynucleotides. In another embodiment, a palindromic sequence comprises a stretch of polynucleotides that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical when read forwards as when read backwards on the complementary strand. In yet another embodiment, a palindromic sequence comprises a stretch of polynucleotides that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical when read forwards as when read backwards on the complementary strand, wherein such stretch is interrupted by one or more stretches of non-palindromic polynucleotides. An ssDNA
encoding one or more palindromic sequences can fold back upon itself, to form double stranded base pairs comprising a secondary structure (e.g., a hairpin loop, or a three-way junction).
1001821 Under appropriate conditions, for example as described in Sections 5.3.3, 5.3.4, and 5.3.5, An IR or an ITR provided in this Section (Section 5.4.1) can fold and form hairpin structures as described in this Section (Section 5.4.1) and Section 5.5, including stems, a primary stem, loops, turning points, bulges, branches, branch loops, internal loops, and/or any combination or permutation of the structural features described in Section 5.5.
1001831 In one embodiment, an IR or ITR for the methods and compositions provided herein comprises one or more palindromic sequences. In some embodiments, an IR
or ITR
described herein comprises palindromic sequences or domains that in addition to forming the primary stem domain can form branched hairpin structures. In some embodiments, an IR or ITR comprises palindromic sequences that can form any number of branched hairpins. In certain specific embodiments, an IR or ITR comprises palindromic sequences that can form 1 to 30, or any subranges of 1 to 30, branched hairpins. In some specific embodiments, an IR
or ITR comprises palindromic sequences that can form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 branched hairpins. In some embodiments, an IR or ITR comprises sequence that can form two branched hairpin structures that lead to a three-way junction domain (T-shaped). In some embodiments, an IR
or ITR comprises sequence that can form three branched hairpin structures that lead to a four-way junction domain (or cruciform structure). In some embodiments, an IR or ITR
comprises sequence that can form a non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. In some embodiments, an IR or ITR comprises sequence that can form interrupted U-shaped hairpin structure including a series of bulges and base pair mismatches. In some embodiments, the branched hairpins all have the same length of stem and/or loop. In some embodiments, one branched hairpin is smaller (e.g. truncated) than the other branched hairpins. Some exemplar embodiments of the hairpin structures and the structural elements of the hairpin structures are depicted in FIG. 1.
1001841 "Hairpin closing base pair- refers to the first base pair following the unpaired loop sequence. Certain stem loop sequences have preferred closing base pairs (e.g. GC in AAV2 ITRs). In one embodiment, the stem loop sequence comprises G-C pair as the closing base pair. In another embodiment, the stem loop sequence comprises C-G pair as the closing base pair.
1001851 "ITR closing base pair" refers to the first and last nucleotide that forms a base pair in a folded ITR. The terminal base pair is usually the pair of nucleotides of the primary stem domain that are most proximal to the non-ITR sequences (e.g. expression cassette) of the DNA molecule. The ITR closing base pair can be any type of base pair (e.g.
CG, AT, GC
or TA). In one embodiment, the ITR closing base pair is a G-C base pair. In another embodiment, the ITR closing base pair is an A-T base pair. In yet another embodiment, the ITR closing base pair is a C-G base pair. In a further embodiment, the ITR
closing base pair is a T-A base pair.
1001861 The disclosure provides that the DNA secondary structure can be computationally predicted according as known and practiced in the art. DNA secondary structures can be represented in several ways- squiggle plot, graph representation, dot-bracket notation, circular plot, arc diagram, mountain plot, dot plot, etc. In circular plots, the backbone is represented by a circle, and the base pairs are symbolized by arcs in the interior of the circle. In arc diagrams, the DNA backbone is drawn as a straight line and the nucleotides of each base pair are connected by an arc. Both circular and arc plots allow for the identification of secondary structure similarities and differences.
1001871 One of the many methods for DNA secondary structure prediction uses the nearest-neighbor model and minimizes the total free energy associated with a DNA structure.
The minimum free energy is estimated by summing individual energy contributions from base pair stacking, hairpins, bulges, internal loops and multi-branch loops.
The energy contributions of these elements are sequence- and length-dependent and have been experimentally determined. The segregation of the sequence into a stem loop and sub-stems can be depicted, for example, by displaying the structure as graph plot. In a linear interaction plot, each residue is represented on the abscissa and semi-elliptical lines connect bases that pair with each other (e.g. FIG. 2A and B).
1001881 In some embodiments, the ITR promotes the long-term survival of the nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITR promotes the permanent survival of the nucleic acid molecule in the nucleus of a cell (e.g., for the entire life-span of the cell). In some embodiments, the ITR promotes the stability of the nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITR inhibits or prevents the degradation of the nucleic acid molecule in the nucleus of a cell.
1001891 In certain embodiments, IRs or ITRs can comprise any viral ITR. In other embodiments, IRs or ITRs can comprise a synthetic palindromic sequence that can form a palindrome hairpin structure that does not expose a 5' or 3' terminus at the outmost apex or turning point of the repeat.
1001901 In some embodiments, the single stranded ITR sequence stretching from one nucleotide of the ITR closing base pair to the other nucleotide of the ITR
closing base pair has a Gibbs free energy (AG) of unfolding under physiological conditions in the range of -10 kcal/mol to -100 kcal/mol. In one embodiment, the Gibbs free energy (AG) of unfolding referred to in the preceding sentence is no more than -10 (meaning <-10, including e.g. -20, -30, etc.), no more than -11, no more than -12, no more than -13, no more than -14, no more than -15, no more than -16, no more than -17, no more than -18, no more than -19, no more than -20, no more than -21, no more than -22, no more than -23, no more than -24, no more than -25, no more than -26, no more than -27, no more than -28, no more than -29, no more than -30, no more than -31, no more than -32, no more than -33, no more than -34, no more than -35, no more than -36, no more than -37, no more than -38, no more than -39, no more than -40, no more than -41, no more than -42, no more than -43, no more than -44, no more than -45, no more than -46, no more than -47, no more than -48, no more than -49, no more than -50, no more than -51, no more than -52, no more than -53, no more than -54, no more than -55, no more than -56, no more than -57, no more than -58, no more than -59, no more than -60, no more than -61, no more than -62, no more than -63, no more than -64, no more than -65, no more than -66, no more than -67, no more than -68, no more than -69, no more than -70, no more than -71, no more than -72, no more than -73, no more than -74, no more than -75, no more than -76, no more than -77, no more than -78, no more than -79, no more than -80, no more than -81, no more than -82, no more than -83, no more than -84, no more than -85, no more than -86, no more than -87, no more than -88, no more than -89, no more than -90, no more than -91, no more than -92, no more than -93, no more than -94, no more than -95, no more than -96, no more than -97, no more than -98, no more than -99, or no more than -100 kcal/mol. In another embodiment, the Gibbs free energy (AG) of unfolding referred to in the preceding sentence is about -10 (meaning <-10, including e.g. -20, -30, etc.), about -11, about -12, about -13, about -14, about -15, about -16, about -17, about -18, about -19, about -20, about -21, about -22, about -23, about -24, about -25, about -26, about -27, about -28, about -29, about -30, about -31, about -32, about -33, about -34, about -35, about -36, about -37, about -38, about -39, about -40, about -41, about -42, about -43, about -44, about -45, about -46, about -47, about -48, about -49, about -50, about -51, about -52, about -53, about -54, about -55, about -56, about -57, about -58, about -59, about -60, about -61, about -62, about -63, about -64, about -65, about -66, about -67, about -68, about -69, about -70, about -71, about -72, about -73, about -74, about -75, about -76, about -77, about -78, about -79, about -80, about -81, about -82, about -83, about -84, about -85, about -86, about -87, about -88, about -89, about -90, about -91, about -92, about -93, about -94, about -95, about -96, about -97, about -98, about -99, or about -100 kcal/mol. In some embodiments, the ITR sequence stretching from one nucleotide of the ITR closing base pair to the other nucleotide of the ITR closing base pair has a Gibbs free energy (AG) of unfolding under physiological conditions in the range of -26 kcal/mol to -95 kcal/mol In some embodiments, the ITR sequence stretching from one nucleotide of the ITR closing base pair to the other nucleotide of the ITR closing base pair contribute to all of the Gibbs free energy (AG) of unfolding for the ITR sequence under physiological conditions.
1001911 In some embodiments, in the folded state, the single stranded IR or ITR has an overall Watson-Crick self-complementarity of approximately 50% to 98%. In one embodiment, in the folded state, the single stranded IR or ITR has an overall Watson-Crick self-complementarity of about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In another embodiment, in the folded state, the single stranded IR or ITR has an overall Watson-Crick self-complementarity of at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, in the folded state, IR or ITR has an overall Watson Crick complementarity of approximately 60% to 98%.

1001921 In some embodiments, the single stranded IR or ITR has an overall GC
content of approximately 60-95%. In certain embodiments, the single stranded IR or ITR
has an overall GC content of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. In other embodiments, the single stranded IR or ITR has an overall GC content of about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%. In some embodiments, the single stranded IR has an overall GC content of approximately 60-91%.
1001931 Table 4 lists the folding free energy, GC content, percent of complementation, length of exemplary ITRs and Table 5 lists the Sequences of the ITRs in Table 4.
Table 4: Folding free energy, GC content, percent of complementation, length of exemplary ITRs.
ITR Length A-T G-C G-T Paired GC AG Compl.
Unpaired Total % kcal/mol %
SEQ ID NO:3 85 8 31 39 79% -83.0 92%
8%
SEQ ID NO:4 77 7 28 35 80% -72.7 91%
9%
SEQ ID NO:5 69 5 26 31 84% -63.6 90%
10%
SEQ ID NO:7 89 7 34 41 83% -90.0 92%
8%
SEQ ID NO:8 71 6 26 32 81% -65.2 90%
10%
SEQ ID NO:9 59 4 22 26 85% -50.7 88%
12%
SEQ ID NO:10 51 2 20 22 91% -41.9 86%
14%
SEQ ID NO:27 70 7 13 20 65% -26.6 57%
43%
SEQ ID NO:29 92 6 18 1 25 75% -52.1 52%
48%
SEQ ID NO:28 102 12 26 38 68% -72.8 75%
25%
SEQ ID NO:31 87 13 23 36 64% -63.0 83%
17%
SEQ ID NO:32 113 18 31 49 63% -93.6 87%

13%
SEQ ID NO:33 83 6 32 38 84% -83.0 92%
8%
SEQ ID NO:34 83 7 31 38 82% -80.0 92%
8%
SEQ ID NO:35 67 6 26 32 81% -79.1 96%
4%
=

Table 5: Sequences of the ITRs in Table 4 SEQ ID NO Sequence SEQ ID NO:3 GCTCGACTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA
CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGTCGAGC
SEQ ID NO:4 GCTCGACTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC
CGGGCTTTGCCCGGGCGGCCTCAGTGAGTCGAGC
SEQ ID NO:5 CGCTGACTCAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG
CTTTGCCCGGGCGGCCTGAGTCAGCG
SEQ ID NO:7 CGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC
GAC GC C C GGGC T TT GC C C GGGC GGC C T CAGTGAGC GAGC GAGC
GCG
SEQ ID NO:8 TCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGG
GCTTTGCCCGGGCGGCCTCAGTGAGCGA
SEQ ID NO :9 ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG
CCCGGGCGGCCTCAGT
SEQ ID NO:10 AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG
GGCGGCCT
SEQ ID NO:27 CCATGCATCCGGCTTTAAACGGGCAACTGCGTCTCATTCACGTT
AGAGACTACAACCGTCGGATGCATGG
SEQ ID NO:28 TTCAAACCTGCCGGGGGAGAAGCGGCGTTTTTTCCCGGCCGCCG
CTTCTCTTCTTCTCCCGCCGCCGGGAAAAAAGGCGGGAGAAGC
CCCGGCAGGTTTGAA
SEQ ID NO :29 GTCCGGGCCATGCTTCAAACCTGCCGGGGCTTCTCCCGCCTTTT
TTCCCGGCGGCGGGAGAAGTAGATTTCTCGTACCTGCATGGCCC
GGAC
SEQ ID NO:31 CCAGCGCTTGGGGTTGACGTGCCACTAAGATCAAGCGGCGCGC
GCGCGCCGCTTGTCTTAGTGTCAAGGCAACCCCAAGCAAGCTG
SEQ ID NO :32 GGTTGACTCTGGGCCAGCTTGCTTGGGGTTGCCTTGACACTAAG
ACAAGCGGCGCGCGCGCGCCGCTTGATCTTAGTGGCACGTCAA
CCCCAAGCGCTGGCCCAGAGTCAACC
SEQ ID NO :33 CGCGCTCGCTCGCTCACTGAGGCCGGGCCAAAGGCCCGACGCC
CGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG
SEQ ID NO:34 CGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC
GACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCG
SEQ ID NO:35 CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCTTTGCCCGGGCG
GC C T CAGT GAGC GAGC GAGC GC G
1001941 The DNA molecules for the methods and compositions provided herein can comprise IR or ITRs of various origins. In one embodiment, the IR or ITR in the DNA
molecule is a viral ITR. "Viral ITR" includes any viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a region comprising a palindrome hairpin structure. In one embodiment, the viral ITR is derived from Parvoviridae.
In another embodiment, the viral ITR derived from Parvoviridae comprises a "minimal required origin of replication" that comprises a viral replication-associated protein binding sequence ("RABS"), which refers to a DNA sequence to which viral DNA
replication-associated proteins ("RAPs") and isoforms thereof, encoded by the Parvoviridae genes Rep and NS1 can bind. In some embodiments the RABS comprises a Rep binding sequence ("RBS") (also referred to as RBE (Rep-binding element)) refers to a nucleotide sequence that includes both the nucleotide sequence recognized by a Rep protein (for replication of viral nucleic acid molecules) and the site of specific interaction between the Rep protein and the nucleotide sequence. In another embodiment, the viral ITR derived from Parvoviridae comprises an RABS which comprises NS1-binding elements ("NSBEs") that replication-associated viral protein NS1 can bind. In some embodiments, viral ITR is derived from Parvoviridae comprises a terminal resolution site ('TRS") at which the viral DNA replication-associated proteins NS1 or Rep can perform an endonucleolytic nick within a sequence at the TRS. and. In yet another embodiment, the viral ITR comprises at least one RBS
or NSBE
and at least one TRS In the context of a virus or recombinant Rep based production of viral genomes, the ITRs mediate replication and virus packaging. As unexpectedly found by the inventors and provided herein, duplex linear DNA vectors with ITRs similar to viral ITRs can be produced without the need for Rep or NS1 proteins and consequently independent of the RABS or TRS sequence for DNA replication. Accordingly, the RABS and TRS can optionally be encoded in the nucleotide sequence disclosed herein but are not required and offer flexibility with regard to designing the ITRs. In one embodiment, the ITR for the methods and compositions provided herein does not comprise RABS. In another embodiment, the ITR for the methods and compositions provided herein does not comprise RBS. In another embodiment, the ITR for the methods and compositions provided herein does not comprise NSBE. In yet another embodiment, the ITR for the methods and compositions provided herein does not comprise TRS. In a further embodiment, the ITR for the methods and compositions provided herein does not comprise either RABS or TRS. In a further embodiment, the ITR for the methods and compositions provided herein comprises RBS, TRS, or both RBS and TRS. In a further embodiment, the ITR for the methods and compositions provided herein comprises NB SE, TRS, or both NB SE and TRS.
1001951 "An ITR pair" refers to two ITRs within a single DNA
molecule. In some embodiments, the two ITRs in the ITR pair are both derived from wild type viral ITRs (e.g.
AAV2 ITR) that have an inverse complement sequence across their entire length.
An ITR
can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring sequence, so long as the changes do not affect the properties and overall three-dimensional structure of the sequence. The disclosure provides that, in some embodiments, the insertion, deletion or substitution of one or more nucleotides can provide the generation of a restriction site for nicking endonuclease without changing the overall three-dimensional structure of the viral ITR. In some aspects, the deviating nucleotides represent conservative sequence changes. In certain embodiments, the sequence of an ITR provided herein can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a restriction site for nicking endonuclease, such that the 3D structures are the same shape in geometrical space. In other embodiments, the sequence of an ITR provided herein can have about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a restriction site for nicking endonuclease, such that the 3D
structures are the same shape in geometrical space.
1001961 .. In some embodiments, a DNA molecule for the methods and compositions provided herein comprises a pair of wt-ITRs. In certain specific embodiments, a DNA
molecule for the methods and compositions provided herein comprises a pair of wt-ITRs selected from the group shown in Table 6. Table 6 shows exemplary ITRs from the same serotype or different serotypes, or different parvoviruses, including AAV
serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV
serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV1 1), or AAV serotype 12 (AAV12); AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome (e.g., NCBI: NC 002077; NC 001401 ; NC001729; NC001829; NC006152; NC 006260; NC
006261), ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), canine, equine, and ovine AAV), ITRs from B19 parvovirus (GenBank Accession No: NC
000883), Minute Virus from Mouse (MVM) (GenBank Accession No. NC 001510);
Goose:
goose parvovirus (GenBank Accession No. NC 001701); snake: snake parvovirus 1 (GenBank Accession No. NC 006148).
Table 6: Exemplary ITR sequences Virus Left ITR Right ITR
(accession number) TCGCTCGCTCGGTGGGGCCTGC CTCGCTCGCTCGGTGGGGCCT
GGACCAAAGGTCCGCAGACGGC GC GGACCAAAGGTCCGCAGAC

Virus Left ITR Right ITR
(accession number) AGAGCTCTGCTCTGCCGGCCCC GGCAGAGCTCTGCTCTGCCGG
ACC GAGC GAGC GAGC GC GC AGA CC C CAC C GAGC GAGC GAGC GC
GAGGGAGTGGGCAA (SEQ ID GCAGAGAGGGAGTGGGCAA
NO:11) (SEQ ID NO:12) TCGCTCGCTCACTGAGGCCGGG CTCGCTCGCTCACTGAGGCCG
CGACCAAAGGTCGCCCGACGCC GGCGACCAAAGGTCGCCCGAC
CGGGCTTTGCCCGGGCGGCCTC GCCCGGGCTTTGCCCGGGCGG
AGTGAGCGAGCGAGCGCGCAGA CCTCAGTGAGCGAGCGAGCGC
GAGGGAGTGGCCAA (SEQ ID GCAGAGAGGGAGT GGCC AA
NO:13) (SEQ ID NO:14) TCGCTCGCTCGGTGGGGCCTGG CTCGCTCGCTCGGTGGGGCCT
CGACCAAAGGTCGCCAGACGGA GGCGACCAAAGGTCGCCAGAC
CGTGCTTTGCACGTCCGGCCCCA GGACGTGCTTTGCACGTCCGG
CC GAGCGAGC GAGTGCGCATAG CCCCACCGAGCGAGCGAGTGC
AGGGAGTGGCCAA (SEQ ID GCATAGAGGGAGTGGCCAA
NO:15) (SEQ ID NO:16) TCGCTCACTCACTCGGCCCTGGA TCGGCCCTGGAGACCAAAGGT
GACCAAAGGTCTCCAGACTGCC CTCCAGACTGCCGGCCTCTGG
GGC C TC TGGC C GGC AGGGC C GA C C GGCAGGGC CGAGT GAGT GA
GTGAGTGAGCGAGCGCGCATAG GCGAGCGCGCATAGAGGGAGT
AGGGAGTGGCCAA (SEQ ID GGCCAA (SEQ ID NO:18) NO:17) (NC 0061 CGCTCGCTGGCTCGTTTGGGGG TCGCTCGCTGGCTCGTTTGGGG
52) GGTGGCAGCTCAAAGAGCTGCC GGGTGGCAGCTCAAAGAGC TG
AGACGACGGCCCTCTGGCCGTC CCAGACGACGGCCCTCTGGCC
GCCCCCCCAAACGAGCCAGCGA GTCGCCCCCCCAAACGAGCCA
GCGAGCGAACGCGACAGGGGG GCGAGCGAGCGAACGCGACAG
GAGAG (SEQ ID NO:19) GGGGGAGAG (SEQ ID NO:20) (NC 0062 TCGCTCGCTCGGTGGGGCCTGC CTCGCTCGCTCGGTGGGGCCT
60) GGACCAAAGGTCCGCAGACGGC GCGGACCAAAGGTCCGCAGAC
AGAGCTCTGCTCTGCCGGCCCC GGCAGAGCTCTGCTCTGCCGG
ACC GAGC GAGC GAGC GC GC ATA CC C CAC C GAGC GAGC GAGC GC
GAGGGAGTGGCCAA (SEQ ID GCATAGAGGGAGTGGCCAA
NO:21) (SEQ ID NO:22) HBOV GTGGTTGTACAGACGCCATCTTG TTGCTTATGCAATCGCGAAACT
(JQ92342 GAATCCAATATGTCTGCCGGCTC CTATATCTTTTAATGTGTTGTT
2) AGTCATGCCTGCGCTGCGCGCA GTTGTACATGCGCCATCTTAGT
GCGCGCTGCGCGCGCGCATGAT TTTATATCAGCTGGCGCCTTAG
CTAATCGCCGGCAGACATATTG TTATATAACATGCATGTTATAT
GATTCCAAGATGGCGTCTGTAC AACTAAGGCGCCAGCTGATAT
AACCAC (SEQ ID NO:23) AAAACTAAGATGGCGCATGTA
CAACAACAACACATTAAAAGA

Virus Left ITR Right ITR
(accession number) TATAGAGTTTCGCGATTGCATA
AGCAA (SEQ ID NO:24) hB19 TGGGCCAGCTTGCTTGGGGTTGC TGGGCCAGCGCTTGGGGTTGA
(AY38633 CTTGACACTAAGACAAGCGGCG CGTGCCACTAAGATCAAGCGG
0) CGCCGCTTGATCTTAGTGGCACG CGCGCCGCTTGTCTTAGTGTCA
TCAACCCCAAGCGCTGGCCCA AGGCAACCCCAAGCAAGCTGG
(SEQ ID NO:25) CCCA (SEQ ID NO:26) 1001971 In some embodiments, the DNA molecules for the methods and compositions provided herein comprise whole or part of the parvoviral genome. The parvoviral genome is linear, 3.9-6.3 kb in size, and the coding region is bracketed by terminal repeats that can fold into hairpin-like structures, which are either different (heterotelomeric, e.g. 1-IBoV) or identical (homotelomeric, e.g. AAV2). In one embodiment, a DNA molecule for the methods and compositions provided herein comprises 2 different ITRs at the 2 ends of the DNA
molecule. In another embodiment, a DNA molecule for the methods and compositions provided herein comprises 2 identical ITRs at the 2 ends of the DNA molecule.
In yet another embodiment, a DNA molecule for the methods and compositions provided herein comprises 2 different ITRs at the 2 ends of the DNA molecule corresponding to the 2 HBoV
ITRs. In a further embodiment, a DNA molecule for the methods and compositions provided herein comprises 2 identical ITRs at the 2 ends of the DNA molecule corresponding to the AAV2 ITR.
1001981 In certain embodiments, the ITR in the DNA molecules provided herein can be an AAV ITR. In other embodiments, the ITR can be a non-AAV ITR. In one embodiment, the ITRs in the DNA molecules provided herein can be derived from an AAV ITR or a non-AAV TR. In some specific embodiments, the ITR can be derived from any one of the family Parvoviridae, which encompasses parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19). In other specific embodiments, the ITR can be derived from the SV40 hairpin that serves as the origin of SV40 replication. Parvoviridae family viruses consist of two subfamilies:
Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. As such, in one embodiment, the ITR can be derived from any one of the subfamily Parvovirinae.
In another embodiment, the ITR can be derived from any one of the subfamily Densovirinae.
1001991 In comparison to the T-shaped AAV ITRs, the human erythrovirus B19 has ITRs that terminate in imperfect, palindromes that can fold into long linear duplexes with a few unpaired nucleotides, creating a series of small, but highly conserved, mismatched bulges. In some embodiments, any parvovirus ITR can be used as an ITR for the DNA
molecules provided herein (e.g. wild type or modified ITR) or can act as a template ITR
for modification and then incorporation in the DNA molecules provided herein. In some specific embodiments, the parvovirus, from which the ITRs of the DNA molecules are derived, is a dependovirus, an erythroparvovirus, or a bocaparvovirus. In other specific embodiments, the ITRs of the DNA molecules provided herein are derived from AAV, B19 or HBoV.
In certain embodiments, the serotype of AAV ITRs chosen for the DNA molecules provided herein can be based upon the tissue tropism of the serotype. AAV2 has a broad tissue tropism, AAV1 preferentially targets to neuronal and skeletal muscle, and AAV5 preferentially targets neuronal, retinal pigmented epithelia, and photoreceptors. A AV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissues AAV9 preferentially targets liver, skeletal and lung tissue. In one embodiment, the ITR or modified ITR of the DNA molecules provided herein is based on an AAV2 ITR. In one embodiment, the ITR or modified ITR of the DNA

molecules provided herein is based on an AAV1 ITR. In one embodiment, the ITR
or modified ITR of the DNA molecules provided herein is based on an AAV5 ITR. In one embodiment, the ITR or modified ITR of the DNA molecules provided herein is based on an AAV6 ITR. In one embodiment, the ITR or modified ITR of the DNA molecules provided herein is based on an AAV8 ITR. In one embodiment, the ITR or modified ITR of the DNA
molecules provided herein is based on an AAV9 ITR.
1002001 In one embodiment, the DNA molecules for the methods and compositions provided herein comprise one or more non-AAV ITR. In a further embodiment, such non-AAV ITR can be derived from hairpin sequences found in the mammalian genome.
In one specific embodiment, such non-AAV ITR can be derived from the hairpin sequences found in the mitochondria] genome including the OriL hairpin sequence (SEQ ID NO:30:
5'CTTCTCCCGCCGCCGGGAAAAAAGGCGGGAGAAGCCCCGGCAGGTTTGAA'3), which adopts a stem-loop structure and is involved in initiating the DNA
synthesis of mitochondria] DNA (see Fuste et al., Molecular Cell, 37, 67-78, January 15, 2010, which is incorporated herein in its entirety by reference). In another specific embodiment, the DNA
molecules for the methods and compositions provided herein comprise an ITR
derived from the OriL sequence that is mirrored to form a T junction with two self-complimentary palindromic regions and a 12-nucleotide loop at either apex of the hairpin. In one embodiment the DNA molecules for the methods and compositions provided herein comprise an ITR derived from the OriL sequence that maintains OriL hairpin loop followed by an unpaired bulge and a GC-rich stem. Some exemplary embodiments of the ITRs derived from mitochondria OriL are depicted in FIG. 2.
1002011 In one embodiment, the DNA molecules for the methods and compositions provided herein comprise one or more non-AAV ITRs that are derived from aptamer. Similar to viral ITRs, aptamers are composed of ssDNA that folds into a three-dimensional structure and have the ability to recognize biological targets with high affinity and specificity. DNA
aptamers can be generated by systematic evolution of ligands by exponential enrichment (SELEX). For example, it has previously been shown that some aptamers can target the nuclei of human cells (See Shen et al ACS Sens. 2019, 4, 6, 1612-1618, which is herein incorporated in its entirety by reference). In one embodiment, the DNA
molecules for the methods and compositions provided herein comprise nucleus targeting aptamer ITRs or their derivatives, wherein the aptamer specifically binds nuclear protein In some embodiments, the aptamer ITRs fold into a secondary structure that can contain such as hairpins as well as internal loops as well bulges and a stem region. Some exemplary embodiments of aptamers or the ITRs derived from are depicted in FIGS. 3A-3C.
1002021 In some specific embodiments, the DNA molecules for the methods and compositions provided herein comprise one or more AAV2 ITR, human erythrovirus ITR goose parvovirus ITR, and/or their derivatives in any combination. In other specific embodiments, the DNA molecules for the methods and compositions provided herein comprise two ITRs selected from AAV2 ITR, human erythrovirus B19 ITR goose parvovirus ITR, and their derivatives, in any combination. In some specific embodiments, the DNA
molecules for the methods and compositions provided herein comprise one or more AAV2 ITR, human erythrovirus B19 ITR goose parvovirus ITR, and/or their derivatives, in any combination, wherein the ITRs remain functional regardless of whether the palindromic regions of their ITRs are in direct, reverse, or any possible combination of 5' and 3' ITR
directionality with respect to the expression cassette (as described in W02019143885, which is herein incorporated in its entirety by reference).
1002031 In some embodiments, a modified IR or ITR in the DNA
molecules provided herein is a synthetic IR sequence that comprises a restriction site for endonuclease such as 5'-GAGTC-3' in addition to various palindromic sequence allowing for hairpin secondary structure formation as described in this Section (Section 5.4.1).
1002041 In certain embodiments, the IR or ITR in the DNA molecules provided herein can be an IR or ITR having various sequence homology with the IR or ITR sequences described in this Section (Section 5.4.1). In other embodiments, the IR or ITR in the DNA molecules provided herein can be an IR or ITR having various sequence homology with the known IR
or ITR sequences of various ITR origins described in this Section (Section 5.4.1) (e.g. viral ITR, mitochondria ITR, artificial or synthetic ITR such as aptamers, etc.). In one embodiment, such homology provided in this paragraph can be a homology of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, such homology provided in this paragraph can be a homology of about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%
1002051 In some embodiments, the IR or ITR in the DNA molecules provided herein can comprise any one or more features described in this Section (Section 5.4.1), in various permutations and combinations.
5.4.2 Restriction Enzymes, Nicking Endonucleases, and Their Respective Restriction Sites; Programmable Nicking Enzymes and Their Targeting Sites 1002061 Various embodiments for the nicking endonucleases, restriction enzymes, and/or their respective restriction sites as describe in Section 5.3.4 are provided for the DNA
molecules provided herein. In some embodiments, the first, second, third, and fourth restriction sites for nicking endonuclease provided for the DNA molecules as described in Section 3 and this Section (Section 5.4) can be all target sequences for the same nicking endonuclease. In some embodiments, the first, second, third, and fourth restriction sites for nicking endonuclease provided for the DNA molecules as described in Section 3 and this Section (Section 5.4) can be target sequences for four different nicking endonucleases. In other embodiments, the first, second, third, and fourth restriction sites for nicking endonucleases are target sequences for two different nicking endonucleases, including all possible combinations of arranging the four sites for two different nicking endonuclease target sequences (e.g. the first restriction site for the first nicking endonuclease and the rest for the second nicking endonuclease, the first and second restriction sites for the first nicking endonuclease and the rest for the second nicking endonuclease, etc.). In certain embodiments, the first, second, third, and fourth restriction sites for nicking endonucleases are target sequences for three different nicking endonucleases, including all possible combinations of arranging the four sites for three different nicking endonuclease target sequences. In some embodiments, the nicking endonuclease and restriction sites for the nicking endonuclease can be any one selected from those described in Section 5.3.4, including Table 2. In further embodiments, each of the first, second, third, and fourth restriction site for nicking endonuclease can be a site for any nicking endonuclease selected from those described in Section 5.3.4, including Table 2.

Table 7 to Table 16 show exemplary modified AAV ITR sequences that harbor two antiparallel recognition sites for the same nicking endonuclease, grouped by nicking endonuclease species. The corresponding alignments for modified sequences of ITRs and wild type of AAV1, AAV2, AAV3, AAV4 left, AAV4 Right, AAV5 and AAV7 are depicted in FIG. 11 to FIG. 17 Table 7: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nb.BvCI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCCCTCAGCGCGCTCGCTCGCT
No:6 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BbvCI; Format: ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
bl AGCGAGCGAGCGCGCTGAGGGGGAGTGGGC
AA
SEQ ID source: AAV1; TTGCCCACTCCCGCTGAGGGCGCTCGCTCGC
No:2 Recogn. Site: TCGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BbvCI; Format: ti ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGCGAGCGAGCGCCCTCAGCGGGAGTGGGC
AA
SEQ ID source: AAV2; TTGGCCACTCCCCCTCAGCGCGCTCGCTCGCT
No:36 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCG
Nb.BbvCI; Format: ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG
bl AGCGAGCGAGCGCGCTGAGGGGGAGTGGCC
AA
SEQ ID source: AAV2; TTGGCCACTCCCGCTGAGGGCGCTCGCTCGC
No:37 Recogn. Site: TCACTGAGGCCGGGCGACCAAAGGTCGCCCG
Nb.BbvCI; Format: ti ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG
AGCGAGCGAGCGCCCTCAGCGGGAGTGGCC
AA
SEQ ID source: AAV3; TTGGCCACTCCCCCTCAGCGCACTCGCTCGCT
No:38 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAG
Nb.BbvCI, Format: ACGGACGTGCTTTGCACGTCCGGCCCCACCG
bl AGCGAGCGAGTGCGCTGAGGGGGAGTGGCC
AA

SEQ ID No: Name Full Sequence SEQ ID source: AAV3; TTGGCC A C TCCCGCTGA GGGC A CTCGCT CGC
No: 39 Recogn. Site: TCGGTGGGGCCTGGCGACCAAAGGTCGCCAG
Nb.BbvCI; Format: ti ACGGACGTGCTTTGCACGTCCGGCCCCACCG
AGCGAGCGAGTGCCCTCAGCGGGAGTGGCC
AA
SEQ ID source: AAV4 left; TTGGCCACTCCCCCTCAGCGCGCTCGCTCACT
No:40 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
N b .BbvC1; Format: CIGCCGGCCICIGGCCGGCAGGGCCGAGIGA
bl GT GAGC GAGC GC GC T GAGGGGGAGT GGC CA

A
SEQ ID source: AAV4 left; TTGGCCACTCCCGCTGAGGGCGCTCGCTCAC
No:41 Recogn. Site: TCACTCGGCCCTGGAGACCAAAGGTCTCCAG
Nb.BbvCI; Format: ti ACTGCCGGCCTCTGGCCGGCAGGGCCGAGTG
AGTGAGCGAGCGCCCTCAGCGGGAGTGGCC
AA
SEQ ID source: AAV4 right; TTGGCCACATTACCTCAGCGCGCTCGCTCACT
No:42 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BbvCI; Format: CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
bl GT GAGC GAGC GC GC T GAGGGGGAGT GGC CA

A
SEQ ID source: AAV4 right; TTGGCCACATTAGCTGAGGGCGCTCGCTCAC
No:43 Recogn. Site: TCACTCGGCCCTGGAGACCAAAGGTCTCCAG
Nb.BbvCI; Format: ti ACTGCCGGCCTCTGGCCGGCAGGGCCGAGTG
AGTGAGCGAGCGCCCTCAGCGGGAGTGGCC
AA
SEQ ID source: AAV5; CTCTCCCCTCAGCCGCGTTCGCTCGCTCGCTG
No :44 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BbvCI; Format: TGCCAGACGACGGCCCTCTCiGCCGTCGCCCC
bl CCCAAACGAGCCAGCGAGCGAGCGAACGCG
GCTGAGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCGCTGAGGCGTTCGCTCGCTCGCTG
No :45 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BbvCI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCC
CCCAAACGAGCCAGCGAGCGAGCGAACGCC
TCAGCGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCCCTCAGCGCGCTCGCTCGCT
No :46 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BbvCI; Format: ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
bl AGC GAGC GAGC GC GC T GAGGGGGAGT GGCC
AA
SEQ ID source: AAV7; TTGGC CAC TCCCGCTGAGGGC GCTCGCT CGC
No:47 Recogn. Site: TCGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BbvCI; Format: ti ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGC GAGC GAGC GC C C T CAGC GGGAGT GGC C
AA

Table 8: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nb.BsmI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCTGAATGCGCGCTCGCTCGCT
No:48 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb BsmT; Format. bl ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGC GAGC GAGC GC GCAT TC AGGGAGT GGGC
AA
SEQ ID source: AAV1; TTGCCCACTCCCTCTCTGCGCATTCGCTCGCT
No :49 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BsmI; Format: tl ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGCGAGCGAATGCGCAGAGAGGGAGTGGGC
AA
SEQ ID source: AAV2; TTGGCCACTCCCTGAATGCGCGCTCGCTCGCT
No:50 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCG
Nb.BsmI; Format: bl ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG
AGC GAGC GAGC GC GCAT TC AGGGAGT GGC C
AA
SEQ ID source: AAV2; TTGGCCACTCCCTCTCTGCGCATTCGCTCGCT
No: 51 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCG
Nb.BsmI; Format: tl ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG
AGCGAGCGAATGCGCAGAGAGGGAGTGGCC
AA
SEQ ID source: AAV3; TTGGCCACTCCCTGAATGCGCACTCGCTCGCT
No: 52 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAG
Nb.BsmI; Format: bl ACGGACGTGCTTTGCACGTCCGGCCCCACCG
AGCGAGCGAGTGCGCATTCAGGGAGTGGCC
AA
SEQ ID source: AAV3; TTGGCCACTCCCTCTATGCGCATTCGCTCGCT
No:53 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAG
Nb.BsmI; Format: tl ACGGACGTGCTTTGCACGTCCGGCCCCACCG
AGCGAGCGAATGCGCATAGAGGGAGTGGCC
AA
SEQ ID source: AAV4 left; TTGGCCACTCCCTGAATGCGCGCTCGCTCACT
No: 54 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb .B smI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATTCAGGGAGTGGCCA
A
SEQ ID source: AAV4 left; TTGGCCACTCCCTCTATGCGCATTCGCTCACT
No: 55 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsmI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAATGCGCATAGAGGGAGTGGCCA
A
SEQ ID source: AAV4 right; TTGGCCACATTAGGAATGCGCGCTCGCTCAC
No: 56 Recogn. Site: TCACTCGGCCCTGGAGACCAAAGGTCTCCAG
Nb.BsmI; Format: bl ACTGCCGGCCTCTGGCCGGCAGGGCCGAGTG

SEQ ID No: Name Full Sequence AGTGAGCGAGCGCGCATTCAGGGAGTGGCC
AA
SEQ ID source: AAV4 right; TTGGCCACATTAGCTATGCGCATTCGCTCACT
No: 57 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsmI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAATGCGCATAGAGGGAGTGGCCA
A
SEQ ID source: AAV5; CTCTCCCCGAATGCGCGTTCGCTCGCTCGCTG
No:58 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BsmI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCC
CCCAAACGAGCCAGCGAGCGAGCGAACGCG
CATTCGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCCCTGTCGCATTCGCTCGCTCGCTG
No:59 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BsmI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCC
CCCAAACGAGCCAGCGAGCGAGCGAATGCG
ACAGGGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCTGAATGCGCGCTCGCTCGCT
No:60 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BsmI; Format: bl ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGCGAGCGAGCGCGCATTCAGGGAGTGGCC
AA
SEQ ID source: AAV7; TTGGCCACTCCCTCTATGCGCATTCGCTCGCT
No:61 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nb.BsmI; Format: ti ACGGCAGAGCTCTGCTCTGCCGGCCCCACCG
AGCGAGCGAATGCGCATAGAGGGAGTGGCC
AA
Table 9: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nb.Bsral SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCGCAATGCGCGCTCGCTCGCT
No:62 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BsrDI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCATTGCGGGAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCCTCATTGCGCGCTCGCTCGCT
No:63 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BsrDI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAATGAGGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTCCCGCAATGCGCGCTCGCTCGCT
No: 64 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BsrDI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCATTGCGGGAGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTCCCTCATTGCGCGCTCGCTCGCT
No: 65 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BsrDI; Format: ti SEQ ID No: Name Full Sequence CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCAATGAGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCGCAATGCGCACTCGCTCGCT
No: 66 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BsrDI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCATTGCGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCTCATTGCGCACTCGCTCGCT
No:67 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BsrDI; Format: tl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCAATGAGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCGCAATGCGCGCTCGCTCACT
No: 68 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsrDI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATTGCGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCTCATTGCGCGCTCGCTCACT
No: 69 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsrDI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAATGAGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCAATGCGCGCTCGCTCACT
No: 70 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsrDI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATTGCGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCATTGCGCGCTCGCTCACT
No: 71 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BsrDI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAATGAGGGAGTGGC CAA
SEQ ID source: AAV5; CTCTCCGCAATGTCGCGTTCGCTCGCTCGCTG
No:72 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BsrDI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGAC
ATTGCGGAGAG
SEQ ID source: AAV5; CTCTCCCCCATTGCGCGTTCGCTCGCTCGCTG
No:73 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BsrDI; Format: tl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGCA
ATGGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCGCAATGCGCGCTCGCTCGCT
No 74 Recogn. Site. CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BsrDI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCATTGCGGGAGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCCTCATTGCGCGCTCGCTCGCT
No: 75 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BsrDI; Format: tl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAATGAGGGAGTGGCCAA

Table 10: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nb.BssSi SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACGAGCTCTCTGCGCGCTCGCTCGCT
No: 76 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BssSI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGAGAGCTCGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCCTCGTGGCGCGCTCGCTCGCT
No: 77 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BssSI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCCACGAGGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACGAGCTCTCTGCGCGCTCGCTCGCT
No:78 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BssSI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCAGAGAGCTCGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTCCCTCGTGGCGCGCTCGCTCGCT
No: 79 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BssSI; Format: tl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCCACGAGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACGAGCTCTATGCGCACTCGCTCGCT
No: 80 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BssSI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGA GCGA GTGCGC A T A GA GCTCGTGGCC A A
SEQ ID source: AAV3; TTGGCCACTCCCTCGTGGCGCACTCGCTCGCT
No: 81 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BssSI; Format: ti CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCCACGAGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACGAGCTCTATGCGCGCTCGCTCACT
No: 82 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BssSI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATAGAGCTCGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCTCGTGGCGCGCTCGCTCACT
No: 83 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BssSI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCCACGAGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACGAGAGCTATGCGCGCTCGCTCACT
No: 84 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BssSI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATAGAGCTCGTGGCCAA
SEQ D source: AAV4 right; TTGGCCACATTCTCGTGGCGCGC,TCGCTCACT
No: 85 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BssSI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCCACGAGGGAGTGGCCAA
SEQ ID source: AAV5; CTCACGAGCCTGTCGCGTTCGCTCGCTCGCTG
No:86 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BssSI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC

SEQ ID No: Name Full Sequence CCAAACGAGCCAGCGAGCGAGCGAACGCGAC
AGGCTCGTGAG
SEQ ID source: AAV5; CTCTCCCTCGTGTCGCGTTCGCTCGCTCGCTG
No: 87 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BssSI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGAC
ACGAGGGAGAG
SEQ ID source: AAV7; TTGGCCACGAGCTCTATGCGCGCTCGCTCGCT
No:88 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BssSI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCATAGAGCTCGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCCTCGTGGCGCGCTCGCTCGCT
No:89 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BssSI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCCACGAGGGAGTGGCCAA
Table 11: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nb.BtsI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCGCAGTGCGCGCTCGCTCGCT
No: 90 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BtsI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCACTGCGGGAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCCTCACTGCGCGCTCGCTCGCT
No: 91 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BtsI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGTGAGGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTCCCGCAGTGCGCGCTCGCTCGCT
No: 92 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BtsI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCACTGCGGGAGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTCCCTCACTGCGCGCTCGCTCGCT
No: 93 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nb.BtsI; Format: ti CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCAGTGAGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCGCAGTGCGCACTCGCTCGCT
No:94 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BtsI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCACTGCGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCTCACTGCGCACTCGCTCGCT
No: 95 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nb.BtsI; Format: ti CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCAGTGAGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCGCAGTGCGCGCTCGCTCACT
No: 96 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BtsI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCACTGCGGGAGTGGCCAA

SEQ ID No: Name Full Sequence SEQ ID source: AAV4 left; TTGGCCACTCCCTCACTGCGCGCTCGCTCACT
No: 97 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BtsI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAGTGAGGGAGTGGC CAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCAGTGCGCGCTCGCTCACT
No:98 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BtsI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCACTGCGGGAGIGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCACTGCGCGCTCGCTCACT
No: 99 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nb.BtsI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAGTGAGGGAGTGGC CAA
SEQ ID source: AAV5; CTCTCCGCAGTGTCGCGTTCGCTCGCTCGCTG
No:100 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BtsI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGAC
ACTGCGGAGAG
SEQ ID source: AAV5; CTCTCCCCACTGCCGCGTTCGCTCGCTCGCTG
No:101 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nb.BtsI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGGC
AGTGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCGCAGTGCGCGCTCGCTCGCT
No:102 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BtsI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCACTGCGGGAGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCCTCACTGCGCGCTCGCTCGCT
No:103 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nb.BtsI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGTGAGGGAGTGGCCAA
Table 12: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nt.AlwI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCTCGATCCGCGCTCGCTCGCT
No:104 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.AlwI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGGATCGAGGGAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTGGATCTCTGCGCGCTCGCTCGCT
No:105 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.AlwI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGAGATCCAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTCCCTCGATCCGCGCTCGCTCGCT
No:106 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.AlwI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGGATCGAGGGAGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTGGATCTCTGCGCGCTCGCTCGCT
No:107 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.AlwI; Format: ti SEQ ID No: Name Full Sequence CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCAGAGATCCAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCTCGATCCGCACTCGCTCGCT
No:108 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.AlwI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGGATCGAGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTGGATCTATGCGCACTCGCTCGCT
No:109 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.AlwI; Format: ti CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCATAGATCCAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCTCGATCCGCGCTCGCTCACT
No:110 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.AlwI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGGATCGAGGGAGTGGC CAA
SEQ ID source: AAV4 left; TTGGCCACTGGATCTATGCGCGCTCGCTCACT
No:111 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.AlwI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATAGATCCAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCGATCCGCGCTCGCTCACT
No:112 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.AlwI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGGATCGAGGGAGTGGC CAA
SEQ ID source: AAV4 right; TTGGCCACAGGATCTATGCGCGCTCGCTCACT
No: 113 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.AlwI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCATAGATCCAGTGGCCAA
SEQ ID source: AAV5; CTCTCCCCCCTGTCGCGATCCCTCGCTCGCTG
No:114 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.AlwI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGGGATCGCGAC
AGGGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCCGGATCGCGTTCGCTCGCTCGCTG
No:115 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.AlwI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGAT
CCGGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCTCGATCCGCGCTCGCTCGCT
No.116 Recogn. Site. CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.AlwI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGGATCGAGGGAGTGG-CCAA
SEQ ID source: AAV7; TTGGCCACTGGATCTATGCGCGCTCGCTCGCT
No:117 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.AlwI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCATAGATCCAGTGGCCAA

Table 13: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nt.BbvCI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCGCTGAGGGCGCTCGCTCGCT
No:118 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BbvCI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCCCTCAGCGGGAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCCCCTCAGCGCGCTCGCTCGCT
No:119 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BbvCI; Format: tl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCTGAGGGGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTCCCGCTGAGGGCGCTCGCTCGCT
No:120 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BbvCI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCCCTCAGCGGGAGTGGCCAA
SEQ D source: AAV2; TTGGCCACTCCCCCTCAGCGCGCTCGCTCGCT
No:121 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BbvCI; Format: tl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCTGAGGGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCGCTGAGGGCACTCGCTCGCT
No:122 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BbvCI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCCCTCAGCGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCCCTCAGCGCACTCGCTCGCT
No:123 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BbvCI; Format: tl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCTGAGGGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCGCTGAGGGCGCTCGCTCACT
No:124 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BbvCI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCCCTCAGCGGGAGTGGCCAA
SEQ ID source: AAV4 left. TTGGCCACTCCCCCTCAGCGCGCTCGCTCACT
No:125 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BbvCI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCTGAGGGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCTGAGGGCGCTCGCTCACT
No:126 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BbvCI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCCCTCAGCGGGAGTGGCCAA
SEQ ID source: AAV4 right; "1"IGGCCACA1IACCICAGCGCGCTCGCTCACT
No:127 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BbvCI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCTGAGGGGGAGTGGCCAA
SEQ ID source: AAV5; CTCTCCCCGCTGAGGCGTTCGCTCGCTCGCTG
No:128 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BbvCI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC

SEQ ID No: Name Full Sequence CCAAACGAGCCAGCGAGCGAGCGAACGCCTC
AGCGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCTCAGCCGCGTTCGCTCGCTCGCTG
No:129 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BbvCI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGGC
TGAGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCGCTGAGGGCGCTCGCTCGCT
No:130 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BbvCI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCCCTCAGCGGGAGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCCCCTCAGCGCGCTCGCTCGCT
No:131 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BbvCI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCTGAGGGGGAGTGGCCAA
Table 14: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nt.BsmAI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTGAGACTCTGCGCGCTCGCTCGCT
No:132 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BsmAI; Format: CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
bl CGAGCGAGCGCGCAGAGTCTCAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCGTCTCTGCGCGCTCGCTCGCT
No:133 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BsmAI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGAGACGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTGAGACTCTGCGCGCTCGCTCGCT
No: 134 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BsmAI; Format: CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
bl CGAGCGAGCGCGCAGAGTCTCAGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTCCGTCTCTGCGCGCTCGCTCGCT
No:135 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BsmAI; Format: ti CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCAGAGACGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTGAGACTATGCGCACTCGCTCGCT
No:136 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BsmAI; Format: CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
bl CGAGCGAGTGCGCATAGTCTCAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCGTCTCTGCGCACTCGCTCGCT
No:137 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BsmAI; Format: ti CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCAGAGACGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTGAGACTATGCGCGCTCGCTCACT
No: 138 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BsmAI; Format: CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
bl GTGAGCGAGCGCGCATAGTCTCAGTGGCCAA

SEQ ID No: Name Full Sequence SEQ ID source: AAV4 left; TTGGCCACTCCGTCTCTGCGCGCTCGCTCACT
No:139 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BsmAI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAGAGACGGAGTGGC CAA
SEQ ID source: AAV4 right; TTGGCCACAGAGACTATGCGCGCTCGCTCACT
No:140 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BsmAI; Format: CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
bl GTGAGCGAGCGCGCATAGICICAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTGTCTCTGCGCGCTCGCTCACT
No: 141 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BsmAI; Format: ti CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCAGAGACGGAGTGGC CAA
SEQ ID source: AAV5; CTCTCCCCCGAGACGCGTTCGCTCGCTCGCTG
No:142 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BsmAI; Format: TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
bl CCAAACGAGCCAGCGAGCGAGCGAACGCGTC
TCGGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCCGTCTCGCGTTCGCTCGCTCGCTG
No:143 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BsmAI; Format: ti TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGAG
ACGGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTGAGACTATGCGCGCTCGCTCGCT
No:144 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BsmAI; Format: CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
bl CGAGCGAGCGCGCATAGTCTCAGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCGTCTCTGCGCGCTCGCTCGCT
No:145 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BsmAI; Format: ti CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCAGAGACGGAGTGGCCAA
Table 15: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nt.BspQI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCGAAGAGCGCGCTCGCTCGCT
No:146 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BspQI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCTCTTCGGGAGTGGGCAA
SEQ ID source: AAV1; TTGCCCACTCCCGCTCTTCGCGCTCGCTCGCTC
No:147 Recogn. Site: GGTGGGGCCTGCGGACCAAAGGTCCGCAGAC
Nt.BspQI; Format: tl GGCAGAGCTCTGCTCTGCCGGCCCCACCGAGC
GAGCGAGCGCGAAGAGCGGGAGTGGGCAA
SEQ ID source: AAV2; TTGGCCACTCCCGAAGAGCGCGCTCGCTCGCT
No:148 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BspQI; Format: bl CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGCTCTTCGGGAGTGGCCAA
SEQ ID source: AAV2; TTGGCCACTCCCGCTCTTCGCGCTCGCTCGCT
No:149 Recogn. Site: CACTGAGGCCGGGCGACCAAAGGTCGCCCGA
Nt.BspQI; Format: ti SEQ ID No: Name Full Sequence CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
CGAGCGAGCGCGAAGAGCGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCGAAGAGCGCACTCGCTCGCT
No:150 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BspQI; Format: bl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGCTCTTCGGGAGTGGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCGCTCTTCGCACTCGCTCGCT
No:151 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BspQI; Format: tl CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
CGAGCGAGTGCGAAGAGCGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCGAAGAGCGCGCTCGCTCACT
No:152 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BspQI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCTCTTCGGGAGTGGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCGCTCTTCGCGCTCGCTCACT
No:153 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BspQI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGAAGAGCGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGAAGAGCGCGCTCGCTCACT
No:154 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BspQI; Format: bl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGCTCTTCGGGAGTGGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCTCTTCGCGCTCGCTCACT
No: 155 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BspQI; Format: tl CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
GTGAGCGAGCGCGAAGAGCGGGAGTGGCCAA
SEQ ID source: AAV5; CTCTCCCGAAGAGCGCGTTCGCTCGCTCGCTG
No:156 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BspQI; Format: bl TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
CCAAACGAGCCAGCGAGCGAGCGAACGCGCT
CTTCGGGAGAG
SEQ ID source: AAV5; CTCTCCCGCTCTTCGCGTTCGCTCGCTCGCTGG
No:157 Recogn. Site: CTCGTTTGGGGGGGTGGCAGCTCAAAGAGCT
Nt.BspQI; Format: tl GCCAGACGACGGCCCTCTGGCCGTCGCCCCCC
CAAACGAGCCAGCGAGCGAGCGAACGCGAAG
AGCGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCGAAGAGCGCGCTCGCTCGCT
No.158 Recogn. Site. CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BspQI; Format: bl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGCTCTTCGGGAGTGGCCAA
SEQ ID source: AAV7; TTGGCCACTCCCGCTCTTCGCGCTCGCTCGCT
No:159 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BspQI; Format: tl CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
CGAGCGAGCGCGAAGAGCGGGAGTGGCCAA

Table 16: Exemplary AAV derived ITRs harboring antiparallel recognition sites for nicking endonuclease Nt.BstNBI:
SEQ ID No: Name Full Sequence SEQ ID source: AAV1; TTGCCCACTCCCTCTCTGCGCGACTCGCTCGC
No: 60 Recogn. Site: TCGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nt.BstNBI; Format: ACGGCAGAGCTCTGCTCTGCCGGCCCCACCGA
bl GC GAGC GAGTC GC GC AGAGAGGGAGT GGGCA
A
SEQ ID source: AAV1; TTGCCGAGTCCCTCTCTGCGCGCTCGCTCGCT
No: 161 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BstNBI; Format: CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
ti C GAGC GAGC GC GC AGAGAGGGACT C GGC AA
SEQ ID source: A AV2; TTGGCCACTCCCTCTCTGCGCGACTCGCTCGC
No:162 Recogn. Site: T C AC TGAGGC C GGGC GAC C AAAGGTC GC
C C G
Nt.BstNBI; Format: ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA
bl GCGAGCGAGTCGCGCAGAGAGGGAGTGGCCA
A
SEQ ID source: AAV2; TTGGCGAGTCCCTCTCTGCGCGCTCGCTCGCT
No: 63 Recogn. Site: C AC T GAGGC C GGGCGAC C AAAGGT C GC
CC GA
Nt.BstNBI; Format: CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
tl CGAGCGAGCGCGCAGAGAGGGACTCGCCAA
SEQ ID source: AAV3; TTGGCCACTCCCTCTATGCGCGACTCGCTCGC
No.164 Recogn Site. TCGGTGGGGCCTGGCGA CC A A A GGTCGCC A G
Nt.BstNBI; Format: ACGGACGTGCTTTGCACGTCCGGCCCCACCGA
bl GCGAGCGAGTCGCGCATAGAGGGAGTGGCCA
A
SEQ ID source: AAV3; TTGGCGAGTCCCTCTATGCGCACTCGCTCGCT
No: 165 Recogn. Site: CGGTGGGGCCTGGCGACCAAAGGTCGCCAGA
Nt.BstNBI; Format: CGGACGTGCTTTGCACGTCCGGCCCCACCGAG
ti CGAGCGAGTGCGCATAGAGGGACTCGCCAA
SEQ ID source: AAV4 left; TTGGCCACTCCCTCTATGCGCGACTCGCTCAC
No: 166 Recogn. Site: TCACTCGGCCCTGGAGACCAAAGGTCTCCAG
Nt.BstNBI; Format: ACTGCCGGCCTCTGGCCGGCAGGGCCGAGTG
bl AGTGAGCGAGTCGCGCATAGAGGGAGTGGCC
AA
SEQ ID source: AAV4 left; TTGGCGAGTCCCTCTATGCGCGCTCGCTCACT
No: 167 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA
Nt.BstNBI; Format: CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
ti GTGAGCGAGCGCGCATAGAGGGACTCGCCAA
SEQ ID source: AAV4 right; TTGGCCACATTAGCTATGCGCGACTCGCTCAC
No:168 Recogn. Site: TCACTCGGCCCTGGAGACCAAAGGTCTCCAG
Nt.BstNBI; Format: ACTGCCGGCCTCTGGCCGGCAGGGCCGAGTG
bl AGTGAGCGAGTCGCGCATAGAGGGAGTGGCC
AA
SEQ ID source: AAV4 right; T TGGC C AGAGT C GC TAT GC GC GC TC
GC T C AC T
No:169 Recogn. Site: CACTCGGCCCTGGAGACCAAAGGTCTCCAGA

SEQ ID No: Name Full Sequence Nt.BstNBI; Format: CTGCCGGCCTCTGGCCGGCAGGGCCGAGTGA
ti GTGAGCGAGCGCGCATAGAGACTCTGGCCAA
SEQ ID source: AAV5; CTCTCCCCCCTGTCGCGACTCGCTCGCTCGCT
No:170 Recogn. Site: GGCTCGTTTGGGGGGGTGGCAGCTCAAAGAG
Nt.BstNBI; Format: CTGCCAGACGACGGCCCTCTGGCCGTCGCCCC
bl CCCAAACGAGCCAGCGAGCGAGCGAGTCGCG
ACAGGGGGGAGAG
SEQ ID source: AAV5; CTCTCCCCCGAGTCGCGTTCGCTCGCTCGCTG
No:171 Recogn. Site: GCTCGTTTGGGGGGGTGGCAGCTCAAAGAGC
Nt.BstNBI; Format: TGCCAGACGACGGCCCTCTGGCCGTCGCCCCC
ti CCAAACGAGCCAGCGAGCGAGCGAACGCGAC
TCGGGGGAGAG
SEQ ID source: AAV7; TTGGCCACTCCCTCTATGCGCGACTCGCTCGC
No:172 Recogn. Site: TCGGTGGGGCCTGCGGACCAAAGGTCCGCAG
Nt.BstNBI; Format: ACGGCAGAGCTCTGCTCTGCCGGCCCCACCGA
bl GCGAGCGAGTCGCGCATAGAGGGAGTGGCCA
A
SEQ ID source: AAV7; TTGGCGAGTCCCTCTATGCGCGCTCGCTCGCT
No:173 Recogn. Site: CGGTGGGGCCTGCGGACCAAAGGTCCGCAGA
Nt.BstNBI; Format: CGGCAGAGCTCTGCTCTGCCGGCCCCACCGAG
tl CGAGCGAGCGCGCATAGAGGGACTCGCCAA
Table 17: Reverse Complement of Nicking Enzyme Targets SEQ ID: Name Sequence AACGGGTGAGGGAGAGACGCGCGAGCGAGCGAGCCACCCCG
186 wt_AAV1 GACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGC
CGGGGTGGCTCGCTC
AACGGGTGAGGGGGAGTCGCGCGAGCGAGCGAGCCACCCCGG
187 AAV1_Nb.BbvCI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCGACTCCCGCGAGCGAGCGAGCCACCCCGG
188 AAV1_Nb.BbvCI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGACTTACGCGCGAGCGAGCGAGCCACCCCGG
189 AAV1_Nb.Bsml_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGAGAGACGCGTAAGCGAGCGAGCCACCCCGG
190 AAV1_Nb.Bsml_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCGTTACGCGCGAGCGAGCGAGCCACCCCGG
191 AAV1_Nb.BsrDI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGAGTAACGCGCGAGCGAGCGAGCCACCCCGG
192 AAV1_Nb.BsrDI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGCTCGAGAGACGCGCGAGCGAGCGAGCCACCCCGG
193 AAV1_Nb.BssSI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC

SEQ ID: Name Sequence AACGGGTGAGGGAGCACCGCGCGAGCGAGCGAGCCACCCCGG
194 AAV1_N b. BssSI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCGTCACGCGCGAGCGAGCGAGCCACCCCGG
195 AAV1_N b. Btsl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGAGTGACGCGCGAGCGAGCGAGCCACCCCGG
196 AAV1_N b. Bts I_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGAGCTAGGCGCGAGCGAGCGAGCCACCCCGG
197 AAV1_Nt.Alwl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGACCTAGAGACGCGCGAGCGAGCGAGCCACCCCGG
198 AAV1_Nt.Alwl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCGACTCCCGCGAGCGAGCGAGCCACCCCGG
199 AAV1_Nt. BbvCI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGGGAGTCGCGCGAGCGAGCGAGCCACCCCGG
200 AAV1_Nt. BbvCI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGACTCTGAGACGCGCGAGCGAGCGAGCCACCCCGG
201 AAV1_Nt. BsmAl_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGCAGAGACGCGCGAGCGAGCGAGCCACCCCGG
202 AAV1_Nt. BsmAl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCTTCTCGCGCGAGCGAGCGAGCCACCCCGG
203 AAV1_Nt. BspQl_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACGGGTGAGGGCGAGAAGCGCGAGCGAGCGAGCCACCCCG
204 AAV1_Nt. BspO.I_BL
GACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGC
CGGGGTGGCTCGCTC
AACGGGTGAGGGAGAGACGCGCTGAGCGAGCGAGCCACCCCG
205 AAV1_Nt. BstN BI_TL
GACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGC
CGGGGTGGCTCGCTC
AACGGCTCAGGGAGAGACGCGCGAGCGAGCGAGCCACCCCGG
206 AAV1_Nt.BstN BI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGAGACGCGCGAGCGAGCGAGTGACTCCGG
207 wt_AAV2 CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGGGAGTCGCGCGAGCGAGCGAGTGACTCCGG
208 AAV2_N b. BbvCI_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCGACTCCCGCGAGCGAGCGAGTGACTCCGG
209 AAV2_N b. BbvCI_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC

SEQ ID: Name Sequence AACCGGTGAGGGACTTACGCGCGAGCGAGCGAGTGACTCCGG
210 AAV2_N b. Bsm I_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGAGACGCGTAAGCGAGCGAGTGACTCCGG
211 AAV2_N b. Bs m I_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCGTTACGCGCGAGCGAGCGAGTGACTCCGG
212 AAV2_N b. BsrDI_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGTAACGCGCGAGCGAGCGAGTGACTCCGG
213 AAV2_N b. BsrDI_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGCTCGAGAGACGCGCGAGCGAGCGAGTGACTCCGG
214 AAV2_N b. BssSI_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGCACCGCGCGAGCGAGCGAGTGACTCCGG
215 AAV2_N b. BssSI_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCGTCACGCGCGAGCGAGCGAGTGACTCCGG
216 AAV2_N b. Btsl_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGTGACGCGCGAGCGAGCGAGTGACTCCGG
217 AAV2_N b. Bts I_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGCTAGGCGCGAGCGAGCGAGTGACTCCGG
218 AAV2_Nt.Alwl_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGACCTAGAGACGCGCGAGCGAGCGAGTGACTCCGG
219 AAV2_Nt.Alwl_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCGACTCCCGCGAGCGAGCGAGTGACTCCGG
220 AAV2_Nt. BbvCI_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGGGAGTCGCGCGAGCGAGCGAGTGACTCCGG
221 AAV2_Nt. BbvCI_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGACTCTGAGACGCGCGAGCGAGCGAGTGACTCCGG
222 AAV2_Nt. BsmAl_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGCAGAGACGCGCGAGCGAGCGAGTGACTCCGG
223 AAV2_Nt. BsmAl_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCTTCTCGCGCGAGCGAGCGAGTGACTCCGG
224 AAV2_Nt. BspQl_TL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGCGAGAAGCGCGAGCGAGCGAGTGACTCCGG
225 AAV2_Nt. BspOl_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC

SEQ ID: Name Sequence AACCGGTGAGGGAGAGACGCGCTGAGCGAGCGAGTGACTCCG
226 AAV2_Nt. BstN BI_TL
GCCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGC
CGGAGTCACTCGCTC
AACCGCTCAGGGAGAGACGCGCGAGCGAGCGAGTGACTCCGG
227 AAV2_Nt.BstN BI_BL
CCCGCTGGTTTCCAGCGGGCTGCGGGCCCGAAACGGGCCCGCC
GGAGTCACTCGCTC
AACCGGTGAGGGAGATACGCGTGAGCGAGCGAGCCACCCCGG
228 wt_AAV3 ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGGGAGTCGCGTGAGCGAGCGAGCCACCCCGG
229 AAV3_N b. BbvCI_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGCGACTCCCGTGAGCGAGCGAGCCACCCCGG
230 AAV3_N b. BbvCI_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGACTTACGCGTGAGCGAGCGAGCCACCCCGG
231 AAV3_N b. Bsm I_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGATACGCGTAAGCGAGCGAGCCACCCCGG
232 AAV3_N b. Bs m I_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGCGTTACGCGTGAGCGAGCGAGCCACCCCGG
233 AAV3_N b. BsrDI_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGTAACGCGTGAGCGAGCGAGCCACCCCGG
234 AAV3_N b. BsrDI_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGCTCGAGATACGCGTGAGCGAGCGAGCCACCCCGG
235 AAV3_N b. BssSI_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGCACCGCGTGAGCGAGCGAGCCACCCCGG
236 AAV3_N b. BssSI_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGCGTCACGCGTGAGCGAGCGAGCCACCCCGG
237 AAV3_N b. Btsl_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGTGACGCGTGAGCGAGCGAGCCACCCCGG
238 AAV3_N b. Bts I_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGCTAGGCGTGAGCGAGCGAGCCACCCCGG
239 AAV3_Nt.Alw I_B L
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGACCTAGATACGCGTGAGCGAGCGAGCCACCCCGGA
240 AAV3_Nt.Alw I_B L
CCGCTGGITTCCAGCGGICTGCCTGCACGAAACGTGCAGGCCG
GGGTGGCTCGCTC
AACCGGTGAGGGCGACTCCCGTGAGCGAGCGAGCCACCCCGG
241 AAV3_Nt. BbvCI_TL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC

SEQ ID: Name Sequence AACCGGTGAGGGGGAGTCGCGTGAGCGAGCGAGCCACCCCGG
242 AAV3_Nt.BbvCI_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGACTCTGATACGCGTGAGCGAGCGAGCCACCCCGGA
243 AAV3_Nt.BsmAl_TL
CCGCTGGITTCCAGCGGICTGCCTGCACGAAACGTGCAGGCCG
GGGTGGCTCGCTC
AACCGGTGAGGCAGAGACGCGTGAGCGAGCGAGCCACCCCGG
244 AAV3_Nt. Bsm ALB L
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGCTTCTCGCGTGAGCGAGCGAGCCACCCCGGA
245 AAV3_Nt.Bsp0.1_TL
CCG CTGGTTTCCAG CGGTCTGCCTGCACGAAACGTGCAGGCCG
GGGTGGCTCGCTC
AACCGGTGAGGGCGAGAAGCGTGAGCGAGCGAGCCACCCCGG
246 AAV3_Nt.BspQl_BL
ACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGATACGCGCTGAGCGAGCGAGCCACCCCG
247 AAV3_Nt.BstN BI_TL
GACCGCTGGTTTCCAGCGGTCTGCCTGCACGAAACGTGCAGGC
CGGGGTGGCTCGCTC
AACCGCTCAGGGAGATACGCGTGAGCGAGCGAGCCACCCCGG
248 AAV3_Nt.BstN BI_BL
ACCGCTGGITTCCAGCGGICTGCCTGCACGAAACGTGCAGGCC
GGGGTGGCTCGCTC
AACCGGTGAGGGAGATACGCGCGAGCGAGTGAGTGAGCCGGG
249 wt_AAV4_I eft ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
CGGCTCACTCACTCGCTCGCGCGTATCTCCCTCACCGGTT
AACCGGTGAGGGGGAGTCGCGCGAGCGAGTGAGTGAGCCGG
AAV4 ¨ left ¨ N b. BbvCI

¨ GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
BL
CCGGCTCACTCACTCGCTCGCGCGACTCCCCCTCACCGGTT
AACCGGTGAGGGCGACTCCCGCGAGCGAGTGAGTGAGCCGGG
AAV4 ¨ left ¨ N b.BbvCI

¨ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
TL
CGGCTCACTCACTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGAGGGACTTACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 ¨ left ¨ N b.Bsm I ¨B

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGTAAGTCCCTCACCGGTT
AACCGGTGAGGGAGATACGCGTAAGCGAGTGAGTGAGCCGGG
AAV4 left N b.Bsm IT

¨ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTTACGCGTATCTCCCTCACCGGTT
AACCGGTGAGGGCGTTACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 ¨ left ¨ N b.Bsr D I ¨B

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGTAACGCCCTCACCGGTT
AACCGGTGAGGGAGTAACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 ¨ left ¨ N b.Bsr D I ¨T

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGTTACTCCCTCACCGGTT
AACCGGTGCTCGAGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 ¨ left ¨ N b.BssS I ¨B

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGTATCTCGAGCACCGGTT
AACCGGTGAGGGAGCACCGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 left N b BssS I ¨T _ _ .

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGGTGCTCCCTCACCGGTT

SEQID: Name Sequence AACCGGTGAGGGCGTCACGCGCGAGCGAGTGAGTGAGCCGGG

AAV4Jeft_NLBtsl_BL ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
CGGCTCACTCACTCGCTCGCGCGTGACGCCCTCACCGGTT
AACCGGTGAGGGAGTGACGCGCGAGCGAGTGAGTGAGCCGG

AAV4Jeft_Nb.Btsl_TL GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
CCGGCTCACTCACTCGCTCGCGCGTCACTCCCTCACCGGTT
AACCGGTGAGGGAGCTAGGCGCGAGCGAGTGAGTGAGCCGG

AAV4Jeft_Nt.Alwl_BL GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
CCGGCTCACTCACTCGCTCGCGCCTAGCTCCCTCACCGGTT
AACCGGTGACCTAGATACGCGCGAGCGAGTGAGTGAGCCGGG

AAV4Jeft_Nt.Alwl_BL ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
CGGCTCACTCACTCGCTCGCGCGTATCTAGGTCACCGGTT
AACCGGTGAGGGCGACTCCCGCGAGCGAGTGAGTGAGCCGGG
AAV4 left NtBbv0 T
262 ¨ ¨
¨ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGAGGGGGAGTCGCGCGAGCGAGTGAGTGAGCCGG
AAV4 left NtBbv0 B
263 ¨ ¨
¨ GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
L
CCGGCTCACTCACTCGCTCGCGCGACTCCCCCTCACCGGTT
AACCGGTGACTCTGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 left NtBsmAl 264 ¨ ¨
¨ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
TL
CGGCTCACTCACTCGCTCGCGCGTATCAGAGTCACCGGTT
AACCGGTGAGGCAGAGACGCGCGAGCGAGTGAGTGAGCCGG
AAV4 left NtBsmAl 265 ¨ ¨
¨ GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
BL
CCGGCTCACTCACTCGCTCGCGCGTCTCTGCCTCACCGGTT
AACCGGTGAGGGCTTCTCGCGCGAGCGAGTGAGTGAGCCGGG
AAV4Jeft_NtBspOl_T

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
L
CGGCTCACTCACTCGCTCGCGCGAGAAGCCCTCACCGGTT
AACCGGTGAGGGCGAGAAGCGCGAGCGAGTGAGTGAGCCGG
AAV4Jeft_NtBspQl_ GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
BL
CCGGCTCACTCACTCGCTCGCGCTTCTCGCCCTCACCGGTT
AACCGGTGAGGGAGATACGCGCTGAGCGAGTGAGTGAGCCGG
AAV4 left NtBstNBI
268 ¨ ¨
¨ GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
TL
CCGGCTCACTCACTCGCTCAGCGCGTATCTCCCTCACCGGTT
AACCGCTCAGGGAGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4 left NtBstNBI

¨ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGTATCTCCCTGAGCGGTT
AACCGGTGTAATCGATACGCGCGAGCGAGTGAGTGAGCCGGG
270 wt_AAV4_Right ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
CGGCTCACTCACTCGCTCGCGCGTATCTCCCTCACCGGTT
AACCGGTGTAATGGAGTCGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nb.BbvCI

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGACTCCCCCTCACCGGTT
AACCGGTGTAATCGACTCCCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nb.BbvCI

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
_TL
CGGCTCACTCACTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGTAATCCTTACGCGCGAGCGAGTGAGTGAGCCGGGA
AAV4_Right_Nb.Bsml ¨ CCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCCC
BL
GGCTCACTCACTCGCTCGCGCGTAAGTCCCTCACCGGTT

SEQ ID: Name Sequence AACCGGTGTAATCGATACGCGTAAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bsm I_ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
TL
CGGCTCACTCACTCGCTTACGCGTATCTCCCTCACCGGTT
AACCGGTGTAATCGTTACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bsr D I

BL
¨
CGGCTCACTCACTCGCTCGCGCGTAACGCCCTCACCGGTT
AACCGGTGTAATCGTAACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bsr D I

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
_TL
CGGCTCACTCACTCGCTCGCGCGTTACTCCCTCACCGGTT
AACCGGTGCTCTCGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bss51 ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGTATCTCGAGCACCGGTT
AACCGGTGTAAGAGCACCGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. BssS I

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
_TL
CGGCTCACTCACTCGCTCGCGCGGTGCTCCCTCACCGGTT
AACCGGIGTAATCGTCACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bts I_ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGTGACGCCCTCACCGGTT
AACCGGTGTAATCGTGACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_N b. Bts I_ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
TL
CGGCTCACTCACTCGCTCGCGCGTCACTCCCTCACCGGTT
AACCGGTGTAATCGCTAGGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.Alw I_ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCCTAGCTCCCTCACCGGTT
AACCGGTGTCCTAGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Rig ht_Nt.Alw I_ ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGTATCTAGGTCACCGGTT
AACCGGTGTAATCGACTCCCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BbvC1 ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
_TL
CGGCTCACTCACTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGTAATGGAGTCGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BbvC1 BL
¨
CGGCTCACTCACTCGCTCGCGCGACTCCCCCTCACCGGTT
AACCGGTGTCTCTGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BsmA I

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
_TL
CGGCTCACTCACTCGCTCGCGCGTATCAGAGTCACCGGTT
AACCGGTGTAACAGAGACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BsmA I

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCGTCTCTGCCTCACCGGTT
AACCGGTGTAATCTTCTCGCGCGAGCGAGTGAGTGAGCCGGGA
AAV4_Right_Nt.BspQ1 CCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCCC
_TL
GGCTCACTCACTCGCTCGCGCGAGAAGCCCTCACCGGTT
AACCGGTGTAATCGAGAAGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BspQ1 ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
BL
CGGCTCACTCACTCGCTCGCGCTTCTCGCCCTCACCGGTT
AACCGGTGTAATCGATACGCGCTGAGCGAGTGAGTGAGCCGG
AAV4_Right_Nt.BstN B

GACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTC
I_TL
CCGGCTCACTCACTCGCTCAGCGCGTATCTCCCTCACCGGTT

SEQ ID: Name Sequence AACCGGTCTCAGCGATACGCGCGAGCGAGTGAGTGAGCCGGG
AAV4_Right_Nt.BstN B

ACCTCTGGTTTCCAGAGGTCTGACGGCCGGAGACCGGCCGTCC
I_BL
CGGCTCACTCACTCGCTCGCGCGTATCTCTGAGACCGGTT
GAGAGGGGGGACAGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
291 wt_AAV5 GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTGTC
CCCCCTCTC
GAGAGGGGAGTCGGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
292 AAV5_N b. BbvCI_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCCGAC
TCCCCTCTC
GAGAGGGGCGACTCCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
293 AAV5_N b. BbvCI_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGGAGTC
GCCCCTCTC
GAGAGGGGCTTACGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
294 AAV5_N b. Bsm I_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCGTAA
GCCCCTCTC
GAGAGGGGGGACAGCGTAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
295 AAVS_N b. Bs m I_TL
GCAGCGGGGGGGITTGCTCGGICGCTCGCTCGCTTACGCTGIC
CCCCCTCTC
GAGAGGCGTTACAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGITTCTCGACGGICTGCTGCCGGGAGACCG
296 AAN/5_N b. BsrDI_BL
GCAGCGGGGGGGITTGCTCGGICGCTCGCTCGCTTGCGCTGTA
ACGCCTCTC
GAGAGGGGGTAACGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
297 AAV5_N b. BsrDI_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCGTTA
CCCCCTCTC
GAGTGCTCGGACAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
298 AAV5_N b. BssSI_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTGTC
CGAGCACTC
GAGAGGGAGCACAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
299 AAVS_N b. BssSI_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTGTG
CTCCCTCTC
GAGAGGCGTCACAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGITTCTCGACGGICTGCTGCCGGGAGACCG
300 AAV5_N b. Btsl_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTGTG
ACGCCTCTC
GAGAGGGGTGACGGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
301 AAV5_N b. Bts I_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCCGTC
ACCCCTCTC
GAGAGGGGGGACAGCGCTAGGGAGCGAGCGACCGAGCAAAC
302 AAV5 Nt.Alwl BL
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG

SEQ ID: Name Sequence GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCCCTAGCGCTGTC
CCCCCTCTC
GAGAGGGGGCCTAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
303 AAVS_Nt.Alwl_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTAGG
CCCCCTCTC
GAGAGGGGCGACTCCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
304 AAV5_Nt. BbvCI_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGGAGTC
GCCCCTCTC
GAGAGGGGAGTCGGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
305 AAV5_Nt. BbvCI_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCCGAC
TCCCCTCTC
GAGAGGGGGCTCTGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
306 AAV5_Nt. BsmAl_TL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCAGAG
CCCCCTCTC
GAGAGGGGGCAGAGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGITTCTCGACGGICTGCTGCCGGGAGACCG
307 AAV5_Nt. BsmAl_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTCTG
CCCCCTCTC
GAGAGGGCTTCTCGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGITTCTCGACGGICTGCTGCCGGGAGACCG
308 AAV5_Nt. BspQl_TL
GCAGCGGGGGGGITTGCTCGGICGCTCGCTCGCTTGCGCGAGA
AG CCCTCTC
GAGAGGGCGAGAAGCGCAAGCGAGCGAGCGACCGAGCAAAC
CCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
309 AAV5_Nt. BspQl_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTTCTC
GCCCTCTC
GAGAGGGGGGACAGCGCTGAGCGAGCGAGCGACCGAGCAAA
CCCCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACC
310 AAV5_Nt. BstN BI_TL
GGCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTCAGCGCTG
TCCCCCCTCTC
GAGAGGGGGCTCAGCGCAAGCGAGCGAGCGACCGAGCAAACC
CCCCCACCGTCGAGTTTCTCGACGGTCTGCTGCCGGGAGACCG
311 AAN/5_Nt.BstN BI_BL
GCAGCGGGGGGGTTTGCTCGGTCGCTCGCTCGCTTGCGCTGAG
CCCCCTCTC
AACCGGTGAGGGAGATACGCGCGAGCGAGCGAGCCACCCCGG
312 wt_AAV7 ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTATCTCCCTCACCGGTT
AACCGGTGAGGGGGAGTCGCGCGAGCGAGCGAGCCACCCCGG
313 AAV7_N b. BbvCI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGIGGCTCGCTCGCTCGCGCGACTCCCCCTCACCGGIT
AACCGGTGAGGGCGACTCCCGCGAGCGAGCGAGCCACCCCGG
314 AAV7_N b. BbvCI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGAGGGACTTACGCGCGAGCGAGCGAGCCACCCCGG
315 AAV7_N b. Bsm I_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGIGGCTCGCTCGCTCGCGCGTAAGTCCCTCACCGGIT

SEQ ID: Name Sequence AACCGGTGAGGGAGATACGCGTAAGCGAGCGAGCCACCCCGG
316 AAV7_N b. Bs m I_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTTACGCGTATCTCCCTCACCGGTT
AACCGGTGAGGGCGTTACGCGCGAGCGAGCGAGCCACCCCGG
317 AAV7_N b. BsrDI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTAACGCCCTCACCGGTT
AACCGGTGAGGGAGTAACGCGCGAGCGAGCGAGCCACCCCGG
318 AAV7_N b. BsrDI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTTACTCCCTCACCGGTT
AACCGGTGCTCGAGATACGCGCGAGCGAGCGAGCCACCCCGG
319 AAV7_N b. BssSI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTATCTCGAGCACCGGTT
AACCGGTGAGGGAGCACCGCGCGAGCGAGCGAGCCACCCCGG
320 AAV7_N b. BssSI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGGTGCTCCCTCACCGGTT
AACCGGTGAGGGCGTCACGCGCGAGCGAGCGAGCCACCCCGG
321 AAV7_N b. Btsl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTGACGCCCTCACCGGTT
AACCGGTGAGGGAGTGACGCGCGAGCGAGCGAGCCACCCCGG
322 AAV7_N b. Bts I_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTCACTCCCTCACCGGTT
AACCGGTGAGGGAGCTAGGCGCGAGCGAGCGAGCCACCCCGG
323 AAV7_Nt.Alwl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCCTAGCTCCCTCACCGGTT
AACCGGTGACCTAGATACGCGCGAGCGAGCGAGCCACCCCGG
324 AAV7_Nt.Alwl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTATCTAGGTCACCGGTT
AACCGGTGAGGGCGACTCCCGCGAGCGAGCGAGCCACCCCGG
325 AAV7_Nt. BbvCI_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGGGAGTCGCCCTCACCGGTT
AACCGGTGAGGGGGAGTCGCGCGAGCGAGCGAGCCACCCCGG
326 AAV7_Nt. BbvCI_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGACTCCCCCTCACCGGTT
AACCGGTGACTCTGATACGCGCGAGCGAGCGAGCCACCCCGGA
327 AAV7_Nt. BsmAl_TL
CGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCCG
GGGTGGCTCGCTCGCTCGCGCGTATCAGAGTCACCGGTT
AACCGGTGAGGCAGAGACGCGCGAGCGAGCGAGCCACCCCGG
328 AAV7_Nt. BsmAl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTCTCTGCCTCACCGGTT
AACCGGTGAGGGCTTCTCGCGCGAGCGAGCGAGCCACCCCGG
329 AAV7_Nt. BspQl_TL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGAGAAGCCCTCACCGGTT
AACCGGTGAGGGCGAGAAGCGCGAGCGAGCGAGCCACCCCGG
330 AAV7_Nt. BspQl_BL
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCTTCTCGCCCTCACCGGTT
AACCGGTGAGGGAGATACGCGCTGAGCGAGCGAGCCACCCCG
331 AAV7_Nt. BstN BI_TL
GACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGC
CGGGGTGGCTCGCTCGCTCAGCGCGTATCTCCCTCACCGGTT

SEQ ID: Name Sequence AACCGCTCAGGGAGATACGCGCGAGCGAGCGAGCCACCCCGG
332 AAV7_Nt.BstN B I_B L
ACGCCTGGTTTCCAGGCGTCTGCCGTCTCGAGACGAGACGGCC
GGGGTGGCTCGCTCGCTCGCGCGTATCTCCCTGAGCGGTT

The first, second, third, and fourth restriction sites for nicking endonuclease can be arranged in various configurations. In some embodiments, the first and the second restriction sites for nicking endonuclease are at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, atleast 96, atleast 97, at least 98, at least 99, atleast 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides apart.
In other embodiments, the first and the second restriction sites for nicking endonuclease are about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides apart.
1002091 Similarly, in certain embodiments, the third and the fourth restriction sites for nicking endonuclease are at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides apart. In further embodiments, the third and the fourth restriction sites for nicking endonuclease are about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70 about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79 about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88 about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides apart.
1002101 The disclosure provides that the overhang described in Sections 3, 5.2 (including 5.3.3), and 5.4 (including 5.4.1) can be the result of the nicking at the first and second restriction sites by nicking endonucleases and denaturing as described in Sections 3 and 5.2 (including 5.3.3). Thus, in some embodiments, the overhang resulted from the nicking at the first and second restriction sites can be the same length as the first and second restriction sites are apart (in number of nucleotides) as described in the preceding paragraphs of this Section (Section 5.4.2). As the nicking endonucleases can cut the DNA within or outside the restriction sites for the nicking endonucleases, in certain embodiments, the overhang resulted from the nicking at the first and second restriction sites can be longer or shorter than the first and second restriction sites are apart by at least 10, at least 11, at least 12, at least 13, at least

14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides. In other embodiments, the overhang resulted from the nicking at the first and second restriction sites can be longer or shorter than the first and second restriction sites are apart by about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides.

Similarly, the disclosure provides that the overhang described in Sections 3, 5.2 (including 5.3.3), and 5.4 (including 5.4.1) can be the result of the nicking at the third and fourth restriction sites by nicking endonucleases and denaturing as described in Sections 3 and 5.2 (including 5.3.3). Thus, in some embodiments, the overhang resulted from the nicking at the third and fourth restriction sites can be the same length as the third and fourth restriction sites are apart (in number of nucleotides) as described in the preceding paragraphs of this Section (Section 5.4.2). As the nicking endonucleases can cut the DNA
within or outside the restriction sites for the nicking endonucleases, in certain embodiments, the overhang resulted from the nicking at the third and fourth restriction sites can be longer or shorter than the third and fourth restriction sites are apart by at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides. In other embodiments, the overhang resulted from the nicking at the third and fourth restriction sites can be longer or shorter than the third and fourth restriction sites are apart by about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides.
1002121 As is clear from the description in Sections 3 and 5.5 and this Section (Section 5.4), the DNA molecules provided herein comprise an expression cassette. In some embodiments, the expression cassette is located between the first and second restriction sites for nicking endonuclease(s) at one end and the third and fourth restriction sites for nicking endonuclease(s) at the other end. In other embodiments, the expression cassette is located within the dsDNA segment of the DNA molecules produced by performing the method steps a to d as described in Sections 3 and 5.2, including the denaturing step described in Section 5.3.3 to provide two ssDNA overhangs. In certain embodiments, the first, second, third, and fourth restriction sites for the nicking endonucleases are arranged such that the length of the dsDNA segment described in this paragraph is at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6, at least kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 1.5kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb. In other embodiments, the first, second, third, and fourth restriction sites for the nicking endonucleases are arranged such that the length of the dsDNA segment described in this paragraph is about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6, about kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.5kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, or about 10 kb.
1002131 As described in Section 5.3.4, incubation with nicking endonucleases will result in a first nick corresponding to the first restriction site for the nicking endonuclease, a second nick corresponding to the second restriction site for the nicking endonuclease, a third nick corresponding to the third restriction site for the nicking endonuclease, and/or a fourth nick corresponding to the fourth restriction site for the nicking endonuclease. The disclosure provides that the first, second, third, and/or fourth nicks can be at various positions relative to the inverted repeat. In one embodiment, the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat. In another embodiment, the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat. In yet another embodiment, the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR

closing base pair of the first inverted repeat. In a further embodiment, the second nick is within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat. In one embodiment, the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat. In another embodiment, the third nick is within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat.
In yet another embodiment, the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat. In a further embodiment, the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat.
In some embodiments, any or any combinations of the first, second, third, and fourth nicks are inside the inverted repeat. In certain embodiments, any or any combinations of the first, second, third, and fourth nicks are outside the inverted repeat. In some additional embodiments, the first, second, third, and fourth nicks can have any relative positions amongst themselves, between any of them and the inverted repeat, and/or between any of them and the expression cassette as described in this Section (Section 5.4.2), in any combination or permutation. In some further embodiments, the first, second, third, and fourth restriction sites for nicking endonucleases can have any relative positions amongst themselves, between any of them and the inverted repeat, and/or between any of them and the expression cassette as described in this Section (Section 5.4.2), in any combination or permutation.
5.4.3 Expression Cassette encoding GDE
1002141 The DNA molecules provided herein may comprise an expression cassette (see also Sections 3, 5.4, and 5.5). An "expression cassette" is a nucleic acid molecule or a part of nucleic acid molecule containing sequences or other information that directs the cellular machinery to make RNA and protein. In some embodiments, an expression cassette comprises a promoter sequence. In certain embodiments, an expression cassette comprises a transcription unit. In yet some other embodiments, an expression cassette comprises a promoter operatively linked to a transcription unit. In one embodiment, the transcription unit comprises an open reading frame (ORF). Embodiments for ORFs for use with the methods and compositions provided herein are further described in the last paragraph of this Section (Section 5.4.3). The expression cassette can further comprise features to direct the cellular machinery to make RNA and protein. In one embodiment, the expression cassette comprises a posttranscriptional regulatory element. In another embodiment, the expression cassette further comprises a polyadenylation and/or termination signal. In yet another embodiment, the expression cassette comprises regulatory elements known and used in the art to regulate (promote, inhibit and/or turn on/off the expression of the ORF). Such regulatory elements include, for example, 5'-untranslated region (UTR), 3'-UTR, or both the 5'UTR
and the 3'UTR In some further embodiments, the expression cassette comprises any one or more features provided in this Section (Section 5.4.3) in any combination or permutation.
1002151 The expression cassette can comprise a protein coding sequence in its ORF (sense strand). Alternatively, the expression cassette can comprise the complementary sequence of the protein coding ORF (anti-sense strand) and the regulatory components and/or other signals for the cellular machinery to produce a sense strand DNA/RNA and the corresponding protein. In some embodiments, the expression cassette comprises a protein sequence without intron. In other embodiments, the expression cassette comprises a protein sequence with intron, which is removed upon transcription and splicing. The expression cassette can also comprise various numbers of ORFs or transcription units. In one embodiment, the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, or 20 ORFs. In another embodiment, the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transcription units.
1002161 The human AGL gene encodes a 1532 amino acid protein (SEQ ID 1;
accession number P35573) with a molecular mass of approximately 174.8 kDa. The AGL gene is located on chromosome 1 at location 1p21.2. AGL is a multifunctional enzyme acting as a 1,4-alpha-D-glucan: 1,4-alpha-D-glucan-4-alpha-D-glycosyltransferase and an amylo-1,6-glucosidase in glycogen degradation and can also be referred to as glycogen debranching enzyme (GDE), glycogen debrancher, amylo-alpha-1,6-glucosidase, 4-alpha-glucanotransferase, EC:2.4.1.25, EC:3.2.1.33. The consensus human AGL coding sequence can be found at NCBI Accession No. NM 000028.2 and translates into SEQ ID NO:
1.

1002171 One of skill in the art will understand that the GDE therapeutic protein includes all splice variants and orthologs of the GDE protein. Essentially any version of the GDE
therapeutic protein or fragment thereof (e.g., functional fragment) can be encoded by and expressed in and from a DNA vector as described herein. GDE therapeutic protein includes intact molecules as well as fragments (e.g., functional) thereof. In some embodiments, the GDE therapeutic protein can be a functional truncated version as outlined in W02020030661A1.
1002181 In some embodiments, the hairpinned DNA molecule for the expression of the GDE protein provide an advantage over traditional AAV vectors, as there is no size constraint for the heterologous nucleic acid sequences encoding a desired protein. Thus, even a full length GDE 4599nt protein can be expressed from a single DNA vector.
Thus, the DNA
vectors described herein can be used to express a therapeutic GDE protein in a subject in need thereof, e g , a subject with a glycogen storage disease Table 18: Exemplary Transgenes Name Sequence GDE MGHSKQIRILLLNEMEKLEKTLFRLEQGYELQFRLGPTLQGKAVTVY
(accession TNYPFPGETFNREKFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYY
number FLQGNEKSGGGYIVVDPILRVGADNHVLPLDCVTLQTFLAKCLGPFDE
P35573) WESRLRVAKESGYNMIHFTPLQTLGL SRSCYSLANQLELNPDF SRPNR
KYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSKWIQEHPECAY
NLVNSPHLKPAWVLDRALWRF SCDVAEGKYKEKGIPALIENDHEIMN
SIRKIIWEDIFPKLKLWEFFQVDVNKAVEQFRRLLTQENRRVTKSDPN

EELNSEKHRLINYHQEQAVNCLLGNVFYERLAGHGPKLGPVTRKHPL
VTRYFTFPFEElDFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFA
EPGSEVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITATY
FQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTGSEDLDN
VFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVGSFVQPCLRPLM
PAIAHALFMDITHDNECPIVHRSAYDALPSTTIVSMACCASGSTRGYD
ELVPHQISVVSEERFYTKWNPEALPSNTGEVNFQSGIIAARCAISKLHQ
ELGAKGFIQVYVDQVDEDIVAVTRHSPSIHQSVVAVSRTAFRNPKTSF
YSKEVPQMCIPGKIEEVVLEARTIERNTKPYRKDENSINGTPDITVEIRE
HIQLNESKIVKQAGVATKGPNEYIQEIEFENLSPGSVIIFRVSLDPHAQV
AVGILRNHLTQFSPHEKSGSLAVDNADPILKIPFASLASRLTLAELNQIL
YRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPKNDLGHP
FCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQAMFFYLKQIPRYLI
PCYFDAILIGAYTTLLDTAWKQMSSFVQNGSTFVKHLSLGSVQLCGV
GKFPSLPILSPALMDVPYRLNEITKEKEQCCVSLAAGLPHFSSGIFRCW
GRDTFIALRGILLITGRYVEARNIILAFAGTLRHGLIPNLLGEGIYARYN
CRDAVWWWLQCIQDYCKMVPNGLDILKCPVSRMYPTDDSAPLPAGT
LDQPLFEVIQEAMQKHMQGIQFRERNAGPQIDRNM_KDEGFNITAGVD
EETGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSAVEIVG
L SKSAVRWLLEL SKKNIFP YHEVTVKRHGKAIK V SYDEWNRKIQDNF

Name Sequence EKLFHVSEDP SDLNEKHPNLVHKRGIYKDSYGAS SPWCDYQLRPNF TI
AMVVAPELF TTEKAWKALEIAEKKLLGPLGMKTLDPDDMVYCGIYD
NALDNDNYNLAKGFNYHQGPEWLWPIGYFLRAKLYFSRLMGPETTA
KTIVLVKNVL SRHYVHLERSPWKGLPELTNENAQYCPF SCET QAW SI
ATILETLYDL (SEQ ID NO: 1) Native ATGGGACACAGTAAACAGATTCGAATTTTACTTCTGAACGAAATG
GDE GAGAAACTGGAAAAGACCCTCTTCAGACTTGAACAAGGGTATGAG
CTACAGTTCCGATTAGGCCCAACTTTACAGGGAAAAGCAGTTACC
GTGTATACAAATTACCCATTTCCTGGAGAAACATTTAATAGAGAA
AAATTCCGTTCTCTGGATTGGGAAAATCCAACAGAAAGAGAAGAT
GATTCTGATAAATACTGTAAACTTAATCTGCAACAATCTGGTTCAT
TTCAGTATTATTTCCTTCAAGGAAATGAGAAAAGTGGTGGAGGTT
ACATAGTTGTGGACCCCATTTTACGTGTTGGTGCTGATAATCATGT
GCTACCCTTGGACTGTGTTACTCTTCAGACATTTTTAGCTAAGTGTT
TGGGACCTTTTGATGAATGGGAAAGCAGACTTAGGGTTGCAAAAG
AATCAGGCTACAACATGATTCATTTTACCCCATTGCAGACTCTTGG
ACTATCTAGGTCATGCTACTCCCTTGCCAATCAGTTAGAATTAAAT
CCTGAC TT TT C AAGAC C TAATAGAAAGTATAC C TGGAAT GAT GT TG
GACAGCTAGTGGAAAAATTAAAAAAGGAATGGAATGTTATTTGTA
TTACTGATGTTGTCTACAATCATACTGCTGCTAATAGTAAATGGAT
CCAGGAACATCCAGAATGTGCCTATAATCTTGTGAATTCTCCACAC
TTAAAACCTGCCTGGGTCTTAGACAGAGCACTTTGGCGTTTCTCCT
GT GAT GTT GC AGAAGGGAAATAC AAAGAAAAGGGAATAC C TGC TT
TGATTGAAAATGATCACCATATGAATTCCATCCGAAAAATAATTTG
GGAGGATATTTTTCCAAAGCTTAAACTCTGGGAATTTTTCCAAGTA
GATGTCAACAAAGCGGTTGAGCAATTTAGAAGACTTCTTACACAA
GAAAATAGGCGAGTAACCAAGTCTGATCCAAACCAACACCTTACG
ATTATTCAAGATCCTGAATACAGACGGTTTGGCTGTACTGTAGATA
TGAACATTGCACTAACGACTTTCATACCACATGACAAGGGGCCAG
CAGCAATTGAAGAATGCTGTAATTGGTTTCATAAAAGAATGGAGG
AATTAAATTCAGAGAAGCATCGACTCATTAACTATCATCAGGAAC
AGGCAGTTAATTGCCTTTTGGGAAATGTGTTTTATGAACGACTGGC
TGGCCATGGTCCAAAACTAGGACCTGTCACTAGAAAGCATCCTTT
AGTTACCAGGTATTTTACTTTCCCATTTGAAGAGATAGACTTCTCC
ATGGAAGAATCTATGATTCATCTGCCAAATAAAGCTTGTTTTCTGA
TGGCACACAATGGATGGGTAATGGGAGATGATCCTCTTCGAAACT
TTGCTGAACCGGGTTCAGAAGTTTACCTAAGGAGAGAACTTATTTG
CTCTGGGAGACAGTGTTAAATTACGCTATGGGAATAAACCAGAGGA
CTGTCCTTATCTCTGGGCACACATGAAAAAATACACTGAAATAACT
GCAACTTATTTCCAGGGAGTACGTCTTGATAACTGCCACTCAACAC
CTCTTCACGTAGCTGAGTACATGTTGGATGCTGCTAGGAATTTGCA
ACCCAATTTATATGTAGTAGCTGAACTGTTCACAGGAAGTGAAGA
TCTGGACAATGTCTTTGTTACTAGACTGGGCATTAGTTCCTTAATA
AGAGAGGCAATGAGTGCATATAATAGTCATGAAGAGGGCAGATTA
GTTTACCGATATGGAGGAGAACCTGTTGGATCCTTTGTTCAGCCCT
GTTTGAGGC,CTTTAATGCCAGCTATTGCACATGCCCTGTTTATGGA
TATTACGCATGATAATGAGTGTCCTATTGTGCATAGATCAGCGTAT
GATGCTCTTCCAAGTACTACAATTGTTTCTATGGCATGTTGTGCTA
GTGGAAGTACAAGAGGCTATGATGAATTAGTGCCTCATCAGATTT

Name Sequence CAGTGGTTTCTGAAGAACGGTTTTACACTAAGTGGAATCCTGAAG
CATTGCCTTCAAACACAGGTGAAGTTAATTTCCAAAGCGGCATTAT
TGCAGCCAGGTGTGCTATCAGTAAACTTCATCAGGAGCTTGGAGC
CAAGGGTTTTATTCAGGTGTATGTGGATCAAGTTGATGAAGACAT
AGTGGCAGTAACAAGACACTCACCTAGCATCCATCAGTCTGTTGT
GGCTGTATCTAGAACTGCTTTCAGGAATCCCAAGACTTCATTTTAC
AGCAAGGAAGTGCCTCAAATGTGCATCCCTGGCAAAATTGAAGAA
GTAGTTCTTGAAGCTAGAACTATTGAGAGAAACACGAAACCTTAT
AGGAAGGATGAGAATTCAATCAATGGAACACCAGATATCACAGTA
GAAATTAGAGAACATATTCAGCTTAATGAAAGTAAAATTGTTAAA
CAAGCTGGAGTTGCCACAAAAGGGCCCAATGAATATATTCAAGAA
ATAGAATTTGAAAACTTGTCTCCAGGAAGTGTTATTATATTCAGAG
TTAGTCTTGATCCACATGCACAAGTCGCTGTTGGAATTCTTCGAAA
TCATCTGACACAATTCAGTCCTCACTTTAAATCTGGCAGCCTAGCT
GTTGACAATGCAGATCCTATATTAAAAATTCCTTTTGCTTCTCTTGC
CTCCAGATTAACTTTGGCTGAGCTAAATCAGATCCTTTACCGATGT
GAATCAGAAGAAAAGGAAGATGGTGGAGGGTGCTATGACATACC
AAACTGGTCAGCCCTTAAATATGCAGGTCTTCAAGGTTTAATGTCT
GTATTGGCAGAAATAAGACCAAAGAATGACTTGGGGCATCCTTTT
TGTAATAATTTGAGATCTGGAGATTGGATGATTGACTATGTCAGTA
ACCGGCTTATTTCACGATCAGGAACTATTGCTGAAGTTGGTAAATG
GTTGCAGGCTATGTTCTTCTACCTGAAGCAGATCCCACGTTACCTT
ATCCCATGTTACTTTGATGCTATATTAATTGGTGCATATACCACTCT
TCTGGATACAGCATGGAAGCAGATGTCAAGCTTTGTTCAGAATGG
TTCAACCTTTGTGAAACACCTTTCATTGGGTTCAGTTCAACTGTGT
GGAGTAGGAAAATTCCCTTCCCTGCCAATTCTTTCACCTGCCCTAA
TGGATGTACCTTATAGGTTAAATGAGATCACAAAAGAAAAGGAGC
AATGTTGTGTTTCTCTAGCTGCAGGCTTACCTCATTTTTCTTCTGGT
ATTTTCCGCTGCTGGGGAAGGGATACTTTTATTGCACTTAGAGGTA
TACTGCTGATTACTGGACGCTATGTAGAAGCCAGGAATATTATTTT
AGCATTTGCGGGTACCCTGAGGCATGGTCTCATTCCTAATCTACTG
GGTGAAGGAATTTATGCCAGATACAATTGTCGGGATGCTGTGTGG
TGGTGGCTGCAGTGTATCCAGGATTACTGTAAAATGGTTCCAAATG
GTCTAGACATTCTCAAGTGCCCAGTTTCCAGAATGTATCCTACAGA
TGATTCTGCTCCTTTGCCTGCTGGCACACTGGATCAGCCATTGTTT
GAAGTCATACAGGAAGCAATGCAAAAACACATGCAGGGCATACA
GTTCCGAGAAAGGAATGCTGGTCCCCAGATAGATCGAAACATGAA
GGACGAAGGTTTTAATATAACTGCAGGAGTTGATGAAGAAACAGG
ATTTGTTTATGGAGGAAATCGTTTCAATTGTGGCACATGGATGGAT
AAAATGGGAGAAAGTGACAGAGCTAGAAACAGAGGAATCCCAGC
CACACCAAGAGATGGGTCTGCTGTGGAAATTGTGGGCCTGAGTAA
ATCTGCTGTTCGCTGGTTGCTGGAATTATCCAAAAAAAATATTTTC
CCTTATCATGAAGTCACAGTAAAAAGACATGGAAAGGCTATAAAG
GTCTCATATGATGAGTGGAACAGAAAAATACAAGACAACTTTGAA
AAGCTATTTCATGTTTCCGAAGACCCTTCAGATTTAAATGAAAAGC
ATCCAAATCTGGTTCACAAACGTGGCATATACAAAGATAGTTATG
GAGCTTCAAGTCCTTGGTGTGACTATCAGCTCAGGCCTAATTTTAC
CATAGCAATGGTTGTGGCCCCTGAGCTCTTTACTACAGAAAAAGC
ATGGAAAGCTTTGGAGATTGCAGAAAAAAAATTGCTTGGTCCCCT

Name Sequence TGGCATGAAAACTTTAGATCCAGATGATATGGTTTACTGTGGAATT
TATGACAATGCATTAGACAATGACAACTACAATCTTGCTAAAGGT
TTCAATTATCACCAAGGACCTGAGTGGCTGTGGCCTATTGGGTATT
TTCTTCGTGCAAAATTATATTTTTCCAGATTGATGGGCCCGGAGAC
TACTGCAAAGACTATAGTTTTGGTTAAAAATGTTCTTTCCCGACAT
TATGTTCATCTTGAGAGATCCCCTTGGAAAGGACTTCCAGAACTGA
C CAATGAGAATGC CC AGTAC TGTC C TT TC AGC TGTGAAACAC AAG
CCTGGTCAATTGCTACTATTCTTGAGACACTTTATGATTTATAG
(SEQ ID NO: 174) Codon ATGGGGCACTCCAAGCAAATTAGGATTCTGCTGTTGAACGAAATG
optimzed GAGAAACTGGAGAAAACCCTGTTCCGATTGGAACAAGGATATGAA
CTGCAATTCCGCCTCGGGCCAACGCTTCAAGGGAAAGCTGTCACC
GT TTACAC CAAT TATCC CTT TC CAGGGGAAACAT TCAATAGAGAG
AAGTTTAGGTCTCTTGATTGGGAGAATCCTACAGAACGGGAAGAT
GACAGTGATAAATAT TGCAAATTGAAT CT TCAACAAAGTGGATCA
T TC CAGTATTAT TT TC TC CAAGGC AACGAAAAGTCAGGAGGAGGG
TACATCGTCGTAGATCCAATTCTGAGAGTGGGTGCCGACAATCAC
GTTCTGCCCCTTGACTGTGTGACACTGCAGACCTTTCTGGC TAAAT
GCCTGGGCCCTTTTGACGAATGGGAATCTCGACTGCGCGTCGCTAA
AGAAAGCGGCTATAACATGATCCATTTTACACC CC TGCAAACCC TT
GGCCTCAGTCGCTCCTGCTACAGCCTGGCAAACCAACTGGAACTT
AATCCTGATTTCTCACGGCCGAATAGGAAGTATACTTGGAACGAC
GTCGGACAACTGGTGGAAAAGCTGAAGAAAGAGTGGAACGTAAT
TTGCATCACCGATGTTGTTTATAACCACACAGCCGCAAACTCTAAA
TGGATACAAGAACATC CCGAGTGC GC CTATAATC TGGTGAACAGC
CCACACCTGAAGCCCGCCTGGGTACTGGATCGCGCTTTGTGGCGGT
TCTCCTGTGACGTTGCCGAAGGTAAATACAAAGAGAAGGGAATAC
CTGCTCTTATTGAGAACGATCATCACATGAACTCCATTCGCAAAAT
TATATGGGAAGATATTTTCCCTAAGCTCAAGCTGTGGGAGTTCTTC
CAAGTGGATGTAAATAAGGCGGTCGAACAATTTAGGCGGCTCCTG
AC GCAGGAAAATC GCAGGGT TAC GAAAAGC GAC C C C AAC C AACA
TCTCACAATTATCCAGGACCCAGAATATCGCAGATTCGGATGCAC
AGTCGATATGAATATTGCGCTGACTACTTTTATTCCC CAC GATAAG
GGC C CC GCTGCTATAGAAGAATGT TGCAAC TGGT TCCATAAGAGA
ATGGAAGAGTTGAACAGCGAAAAGCACAGGCTCATCAATTATCAC
CAAGAGCAAGCCGTTAACTGTCTCCTTGGTAATGTATTCTATGAAC
GCCTCGCTGGACATGGACCCAAACTCGGGCCCGTGACCAGGAAAC
ACCCACTTGTTACGCGATACTTCACCTTCCCCTTTGA AGA A ATCGA
CTTCTCAATGGAAGAGAGCATGATTCATTTGCCAAATAAAGCCTG
CTTTCTGATGGCTCATAACGGATGGGTTATGGGAGATGACCCCCTG
AGAAATTTTGCTGAAC CAGGC TCC GAAGTTTATC TGC GCCGCGAGT
TGAT ATGT TGGGGAGACAGCGTGAAACTCCGAT AT GGCAACAAGC
CTGAAGATTGCCCGTATCTGTGGGCACATATGAAGAAGTATACTG
AAAT TACTGCGACC TAT TT TCAAGGTGTGAGACTGGATAATTGC CA
TTCCACCCCACTTCATGTGGCCGAATACATGTTGGATGCTGCACGA
AACCTGCAACCAAATCTGTACGTCGTGGCAGAATTGTTCACAGGG
TC CGAGGAC CT TGATAACGTGTTCGTCACCAGATTGGGAATAAGC
TC CC TTATC CGCGAGGCTATGAGTGC TTATAACTCACATGAGGAAG
GAC GCTTGGTGTATAGATAC GGCGGAGAAC CAGTCGGC TC AT T TG

Name Sequence TACAACCCTGTTTGAGACCTCTTATGCCCGCCATAGCACATGCTCT
CTTTATGGATATTACCCATGACAATGAATGTCCTATCGTCCACAGG
TCCGCATATGATGCCCTGCCCAGTACCACAATTGTGAGTATGGCCT
GCTGCGCCTCAGGGTCAACACGCGGTTACGATGAGCTGGTGCCTC
ACCAAATCTCTGTAGTGTCAGAGGAACGCTTCTACACCAAATGGA
ATCCAGAAGCTCTCCCTTCTAACACAGGCGAGGTAAATTTTCAATC
AGGGATAATTGCTGCGCGGTGTGCCATCAGTAAATTGCATCAGGA
GCTGGGAGCTAAAGGCTTTATTCAGGTATACGTCGACCAGGTAGA
CGAAGATATTGTGGCTGTCACTCGCCATAGTCCAAGCATTCACCAG
AGCGTTGTCGCGGTTTCCAGGACAGCTTTCCGCAACCCCAAGACCT
CATTTTACTCAAAAGAGGTGCCACAAATGTGTATACCTGGGAAAA
TAGAGGAAGTAGTCCTGGAGGCACGGACAATAGAAAGAAACACA
AAACCCTATCGCAAGGATGAGAACTCAATAAACGGCACGCCCGAT
ATTACGGTGGAGATACGCGAGCATATTCAGCTGAATGAATCTAAG
ATTGTTAAGCAAGCAGGTGTCGCGACAAAGGGACCTAATGAATAC
ATCCAGGAGATTGAGTTCGAGAACTTGTCCCCAGGAAGCGTGATC
ATCTTCAGGGTGAGCCTCGATCCTCACGCTCAAGTTGCTGTCGGCA
TCCTCAGAAATCACCTGACGCAATTTAGCCCACACTTCAAATCAGG
CTCTCTTGCTGTCGATAATGCTGACCCCATTCTCAAAATTCCCTTTG
CTTCCCTGGCGTCTCGACTGACGCTGGCAGAACTGAATCAGATCCT
GTACAGGTGTGAAAGTGAGGAAAAGGAAGACGGCGGCGGTTGCT
ATGATATACCCAACTGGTCTGCCCTCAAATACGCTGGGCTCCAGG
GGCTGATGTCCGTGCTCGCGGAGATCCGCCCCAAGAACGACCTGG
GGCACCCATTCTGTAATAATCTCCGCAGTGGCGACTGGATGATCG
ATTACGTCTCCAATCGCCTCATCAGCAGAAGCGGTACAATCGCGG
AAGTCGGAAAATGGCTTCAAGCTATGTTCTTTTACCTGAAGCAAAT
TCCCAGGTATCTCATCCCATGTTACTTCGATGCTATATTGATCGGA
GCGTACACAACCCTCTTGGATACCGCCTGGAAACAGATGTCTAGTT
TTGTCCAAAACGGATCTACATTCGTGAAGCACCTCTCACTGGGGTC
CGTGCAGCTTTGTGGGGTCGGGAAATTTCCCAGCTTGCCGATTCTC
TCTCCAGCCCTCATGGATGTCCCCTATCGGCTCAACGAGATTACCA
AGGAGAAAGAGCAGTGCTGCGTTAGCCTGGCCGCTGGACTTCCGC
ATTTCTCTAGCGGGATTTTCCGATGTTGGGGCAGAGACACCTTCAT
AGCTCTCAGGGGCATTCTGCTTATTACAGGTCGCTACGTCGAAGCC
CGCAACATCATTCTGGCTTTTGCAGGAACTTTGCGGCACGGCCTCA
TACCAAATCTCCTCGGCGAGGGGATCTACGCGAGGTACAATTGTC
GAGACGCGGTCTGGTGGTGGCTTCAATGTATACAAGACTACTGTA
AAATGGTTCCGAACGGGCTGGACATACTGAAATGTCCAGTCTCCC
GCATGTACCCGACAGATGATTCTGCTCCACTTCCTGCTGGGACCCT
CGATCAGCCTCTCTTCGAAGTAATACAAGAGGCTATGCAAAAGCA
CATGCAAGGCATTCAGTTCAGGGAGCGCAACGCAGGCCCACAAAT
TGACAGGAACATGAAAGACGAAGGCTTTAACATCACCGCTGGTGT
TGATGAAGAGACAGGCTTTGTATACGGCGGAAATCGCTTCAACTG
CGGGACCTGGATGGACAAGATGGGCGAATCTGATAGGGCTCGCAA
CAGAGGCATCCCCGCGACACCACGGGATGGTAGTGCAGTAGAAAT
CGTTGGGCTTTCTAAATCCGCCGTACGCTGGCTTCTGGAACTCAGT
AAGAAGAACATCTTTCCCTACCACGAAGTCACAGTTAAACGCCAC
GGCAAAGCTATCAAAGTCTCATACGACGAATGGAATAGGAAGATC
CAAGACAACTTCGAGAAGCTCTTTCACGTGAGCGAGGACCCAAGT

Name Sequence GATCTGAATGAAAAGCACCCTAATCTTGTTCATAAGCGAGGCATC
TATAAAGATAGCTACGGGGCTTCAAGTCCCTGGTGTGACTACCAA
CTTAGACCCAACTTCACAATCGCTATGGTGGTAGCCCCCGAGCTCT
TTACGACAGAGAAGGCTTGGAAAGCATTGGAAATCGCCGAGAAG
AAGCTCTTGGGCCCCTTGGGAATGAAAACACTGGACCCTGACGAT
ATGGTTTATTGTGGCATTTATGACAATGCACTCGATAATGACAATT
ATAACTTGGCAAAGGGTTTTAATTACCACCAAGGTCCCGAATGGC
TGTGGCCCATTGGATACTTCTTGCGAGCTAAACTGTATTTCTCCAG
ACTTATGGGACCCGAGACCACAGCTAAGACCATCGTTTTGGTTAA
GAACGTCCTGTCCAGACACTATGTTCACTTGGAGAGAAGTCCTTGG
AAAGGGCTGCCCGAACTGACCAATGAAAACGCACAATACTGTCCC
TTCAGCTGTGAAACACAAGCGTGGTCAATCGCTACAATCCTGGAA
ACTCTGTACGATCTCTGA (SEQ ID NO: 175) Optimized ATGGGCCATAGCAAACAAATACGCATACTGCTGCTCAATGAGATG
Construct GAGAAACTTGAGAAAACACTGTTTCGCCTGGAGCAGGGATACGAA
1 in CTTCAATTTAGATTGGGACCTACCCTTCAAGGGAAGGCCGTGACTG
examples TTTACACTAACTATCCTTTCCCCGGTGAGACCTTCAACCGGGAGAA
GTTTCGGAGCTTGGACTGGGAGAACCCCACTGAGCGAGAGGACGA
CAGTGACAAGTATTGCAAGCTGAACCTTCAGCAGTCCGGGAGTTT
CCAATACTACTTTCTCCAGGGTAACGAAAAGTCTGGCGGTGGCTAT
ATTGTCGTCGATCCTATACTGAGGGTCGGGGCAGACAACCACGTT
CTGCCGCTCGATTGCGTCACGCTGCAAACGTTCTTGGCAAAATGCC
TTGGGCCCTTCGACGAGTGGGAGAGCCGGCTCCGTGTCGCTAAAG
AGAGTGGTTATAATATGATCCACTTCACTCCTCTGCAAACCCTGGG
GCTCAGCAGATCCTGTTATAGCCTGGCAAACCAACTTGAGCTGAA
CCCCGATTTCTCCAGGCCCAACCGTAAATACACTTGGAACGACGT
GGGGCAACTTGTCGAGAAGCTGAAGAAAGAGTGGAACGTCATCTG
CATCACCGACGTGGTGTATAACCACACAGCCGCCAACTCCAAGTG
GATTCAAGAGCACCCCGAGTGCGCGTACAACCTGGTCAACTCACC
GCATCTTAAGCCGGCTTGGGTGCTGGATCGGGCTCTGTGGAGATTT
TCTTGCGACGTGGCTGAGGGTAAGTACAAGGAGAAAGGGATCCCA
GCGCTGATCGAGAACGACCATCACATGAACTCTATTCGCAAGATT
ATATGGGAAGACATCTTCCCGAAACTGAAGCTGTGGGAGTTCTTTC
AGGTGGACGTGAATAAGGCCGTAGAACAGTTCAGGCGGTTGCTGA
CCCAGGAGAACAGAAGGGTGACGAAAAGCGACCCCAATCAGCAT
CTCACTATAATCCAGGACCCCGAGTATCGGCGATTCGGGTGCACC
GTTGACATGAATATAGCTCTCACAACATTTATTCCCCACGATAAAG
GACCGGCCGCTATAGAGGAGTGTTGCA ACTGGTTCCA CA A GCGGA
TGGAAGAGCTGAACTCCGAAAAGCACCGCCTTATCAATTACCACC
AAGAGCAAGCCGTGAACTGTCTGCTCGGGAACGTCTTCTACGAGA
GGCTCGCCGGGCACGGCCCGAAGCTGGGCCCAGTTACCCGCAAAC
ACCCACTGGTGACTAGGTACTTCACCTTTCCCTTCGAGGAAATCGA
TTTTAGCATGGAAGAGAGTATGATCCATCTCCCCAACAAGGCGTG
CTTCCTCATGGCCCATAACGGCTGGGTGATGGGCGACGACCCGTT
GCGTAATTTCGCGGAGCCAGGAAGCGAGGTCTATCTGCGGCGCGA
GCTCATCTGTTGGGGAGATTCCGTGAAACTTCGATACGGAAACAA
GCCCGAAGATTGCCCCTACCTGTGGGCTCATATGAAGAAGTATAC
CGAGATTACCGCTACATACTTTCAAGGCGTTAGGTTGGACAATTGT
CATTCTACCCCGTTGCATGTGGCCGAATATATGCTCGACGCCGCCA

Name Sequence GAAACCTGCAACCAAACCTGTACGTGGTGGCAGAGCTCTTTACTG
GGTCAGAGGACTTGGATAACGTGTTCGTCACACGACTTGGGATAT
CAAGTCTTATTCGGGAAGCTATGTCTGCCTACAACTCCCACGAGGA
AGGACGCCTGGTGTATCGTTACGGTGGGGAGCCCGTGGGGAGTTT
CGTGCAACCATGCCTCAGGCCTCTGATGCCTGCCATCGCGCACGCA
CTTTTCATGGACATCACTCACGACAACGAATGCCCCATAGTTCACA
GGAGTGCCTACGACGCCCTGCCTTCAACAACCATCGTCAGCATGG
CCTGCTGCGCCAGTGGCAGCACTCGCGGGTACGACGAGCTGGTCC
CACACCAAATCAGCGTTGTCTCCGAGGAGAGATTCTATACCAAAT
GGAACCCGGAAGCCCTGCCCTCTAATACTGGAGAGGTGAACTTTC
AGAGTGGGATCATCGCTGCACGGTGCGCAATTTCCAAGTTGCACC
AAGAACTCGGCGCAAAAGGATTCATCCAAGTATACGTCGACCAGG
TGGACGAGGATATCGTTGCCGTTACCCGTCATTCCCCAAGTATTCA
CCAATCCGTCGTAGCAGTTTCACGCACCGCATTTCGGAACCCAAA
GACCAGTTTCTATTCCAAAGAGGTTCCGCAGATGTGTATTCCCGGG
AAGATCGAGGAAGTCGTACTCGAAGCACGAACAATCGAACGAAA
TACTAAGCCATACCGTAAAGACGAAAACTCCATTAACGGCACCCC
TGACATAACCGTGGAGATCCGCGAGCACATACAACTCAACGAGAG
CAAGATCGTGAAGCAGGCAGGGGTGGCGACTAAGGGACCTAACG
AGTACATCCAGGAGATCGAGTTCGAGAATCTGAGCCCCGGTTCAG
TCATAATTTTCCGAGTGTCCTTGGACCCCCACGCCCAGGTGGCAGT
GGGCATCCTGCGGAACCACTTGACGCAGTTTTCTCCCCATTTCAAG
AGTGGGTCCCTGGCCGTGGATAACGCTGACCCCATCCTTAAGATCC
CCTTCGCCAGTTTGGCAAGTCGCCTGACCCTTGCGGAACTCAACCA
AATTTTGTATAGATGCGAGAGTGAGGAGAAAGAGGACGGCGGCG
GATGTTACGATATCCCTAATTGGAGTGCACTGAAGTACGCCGGGTT
GCAGGGGCTTATGAGTGTCCTTGCTGAGATCCGTCCCAAGAACGA
TCTTGGTCACCCCTTCTGCAACAACCTGAGGAGCGGTGACTGGATG
ATCGATTACGTATCTAATAGACTGATAAGTAGGTCCGGCACGATA
GCCGAGGTGGGCAAGTGGCTGCAAGCCATGTTCTTTTATTTGAAAC
AAATTCCCAGATATTTGATTCCTTGCTATTTCGACGCCATCCTGAT
CGGAGCGTACACGACACTGTTGGACACTGCCTGGAAACAAATGTC
CAGTTTCGTGCAAAACGGGTCTACATTCGTTAAGCATTTGAGCCTG
GGGAGCGTACAGCTCTGCGGCGTCGGGAAGTTTCCCTCACTTCCTA
TACTGTCTCCAGCACTGATGGACGTGCCCTACCGTCTGAACGAAAT
TACCAAGGAGAAAGAACAGTGCTGCGTCAGCCTCGCAGCCGGGCT
CCCCCACTTCTCTTCCGGAATATTTCGGTGTTGGGGACGCGACACA
TTCATCGCTCTCCGCGGCATCCTCTTGATCACGGGGAGATACGTGG
AAGCTCGGAACATAATATTGGCCTTCGCCGGAACGCTTAGACACG
GCCTTATACCCAACCTGTTGGGCGAGGGCATCTACGCTCGTTATAA
CTGCCGCGACGCCGTCTGGTGGTGGCTTCAATGCATTCAAGACTAT
TGCAAGATGGTGCCCAACGGGCTGGATATCCTGAAATGTCCTGTG
TCACGGATGTACCCCACCGACGACAGCGCCCCACTCCCGGCCGGG
ACGCTCGACCAACCTCTGTTCGAGGTGATCCAAGAGGCCATGCAG
AAGCATATGCAAGGAATCCAATTTCGTGAGCGCAACGCCGGACCA
CAAATCGACCGCAATATGAAAGATGAGGGGTTCAACATCACAGCC
GGTGTCGACGAGGAGACGGGCTTCGTGTACGGTGGCAACAGGTTT
AACTGCGGGACTTGGATGGACAAGATGGGCGAGAGTGATCGAGC
GAGGAATCGAGGCATTCCCGCTACCCCACGCGACGGCAGCGCTGT

Name Sequence CGAGATCGTTGGGCTCTCAAAGTCCGCGGTCAGGTGGCTGTTGGA
GCTGTCTAAGAAGAACATCTTTCCCTACCACGAGGTAACGGTCAA
GAGGCACGGTAAAGCCATCAAAGTGAGCTACGACGAATGGAATC
GTAAGATTCAGGATAATTTCGAGAAACTCTTCCACGTATCTGAGG
ATCCATCCGACCTCAACGAGAAACACCCCAACTTGGTGCATAAGA
GAGGGATTTATAAGGACAGTTACGGCGCCTCTAGCCCCTGGTGCG
ATTACCAAC TGAGACC C AAC TTC ACAATCGC CATGGTC GTC GC TC C
AGAATTGTTCACCACTGAGAAGGCCTGGAAGGCACTGGAAATCGC
GGAGAAGAAGCTGTTGGGGCCACTCGGTATGAAGACGCTGGACCC
GGACGACATGGTGTATTGCGGTATCTACGATAACGCCTTGGATAA
CGATAATTATAACCTCGCAAAGGGCTTTAACTACCATCAGGGCCC
CGAATGGCTTTGGCCGATAGGTTACTTCTTGCGCGCCAAACTTTAC
TTCTCTAGGCTGATGGGACCCGAAACAACCGCCAAAACAATCGTA
CTCGTGAAGAACGTGTTGAGTAGGCACTACGTGCACCTCGAAAGG
AGCCCATGGAAGGGGCTGCCTGAGCTCACAAACGAAAACGCACA
ATATTGCCCCTTTTCATGCGAGACCCAGGCATGGAGCATCGCCACC
ATACTGGAAACCCTGTACGACTTGTGA (SEQ ID NO: 178) GDE cpg ATGGGTCACTCTAAACAGATAAGAATCCTCCTCCTCAATGAGATG
minimized GAAAAACTTGAAAAAACTCTCTTTAGATTGGAGCAAGGTTATGAG
CTCCAATTTAGATTGGGTCCAACTCTCCAAGGAAAAGCTGTAACTG
TATATACAAATTATCCTTTTCCTGGAGAAACATTTAATAGAGAAAA
ATTTAGATCATTGGATTGGGAAAATCCAACTGAAAGAGAAGATGA
TAGTGATAAGTACTGTAAGTTGAACCTCCAACAAAGTGGTAGTTTT
CAGTATTATTTTCTCCAAGGAAATGAAAAATCTGGAGGAGGATAT
ATTGTAGTGGACCCCATACTTAGAGTTGGTGCAGATAACCATGTTC
TCCCTCTGGATTGTGTAACTTTGCAAACATTTTTGGCCAAATGTCT
GGGTCCTTTTGATGAATGGGAATCAAGATTGAGGGTTGCTAAAGA
ATCTGGATATAATATGATCCATTTTACACCCTTGCAGACATTGGGT
CTGTCAAGGTCTTGTTATTCACTTGCTAATCAACTGGAACTGAATC
CAGATTTTTCAAGACCTAATAGGAAGTATACATGGAATGATGTTG
GACAACTTGTAGAAAAATTGAAGAAAGAATGGAATGTTATTTGCA
TAACTGATGTAGTCTATAATCATACAGCAGCTAATAGTAAATGGA
TACAAGAACATCCTGAATGTGCATATAATTTGGTTAATTCTCCACA
TCTTAAACCAGCATGGGTTTTGGATAGAGCCCTGTGGAGGTTTTCA
TGTGATGTTGCAGAAGGAAAATATAAAGAAAAAGGTATTCCAGCA
CTTATTGAAAATGATCATCATATGAATAGTATCAGAAAGATTATTT
GGGAAGACATATTTCCTAAGTTGAAATTGTGGGAATTTTTTCAAGT
GGATGTTAACAAAGCAGTTGAACAATTCAGAAGACTTCTCACACA
AGAAAATAGAAGAGTAACCAAATCAGATCCTAATCAACATCTTAC
TATCATACAAGATCCTGAATATAGAAGATTTGGTTGTACAGTAGA
CATGAATATTGCTCTCACTACTTTTATACCACATGATAAAGGTCCA
GCTGCAATAGAAGAATGTTGTAATTGGTTTCATAAGAGAATGGAA
GAATTGAATAGTGAAAAACATAGATTGATAAATTATCATCAAGAA
CAAGCTGTAAACTGCTTGTTGGGAAATGTATTCTATGAAAGACTTG
CAGGTCATGGACCAAAATTGGGTCCAGTAACTAGAAAACATCCAT
TGGTTACTAGATATTTTACATTTCCATTTGAAGAAATTGATTTTAGT
ATGGAAGAATCAATGATTCATCTCCCTAATAAAGCCTGTTTTTTGA
TGGCACATAATGGATGGGTTATGGGAGATGATCCTCTTAGAAATTT
TGCAGAACCAGGAAGTGAAGTTTATTTGAGAAGAGAACTTATATG

Name Sequence TTGGGGTGATTCAGTTAAATTGAGATATGGCAATAAACCAGAAGA
TTGTCCATATCTTTGGGCACATATGAAAAAGTATACTGAAATTACT
GCAACATATTTCCAAGGAGTTAGATTGGATAATTGTCATTCTACAC
CTCTCCATGTTGCAGAATATATGCTGGATGCTGCTAGAAATCTTCA
ACCTAATTTGTATGTAGTTGCAGAATTGTTTACTGGATCTGAAGAT
TTGGATAATGTCTTTGTTACAAGATTGGGTATCAGTAGCTTGATAA
GAGAAGCTATGTCAGCATATAATTCTCATGAAGAAGGTAGATTGG
TATATAGATATGGAGGAGAACCAGTTGGTAGTTTTGTTCAACCTTG
TTTGAGACCACTTATGCCAGCAATTGCTCATGCACTCTTTATGGAT
ATTACACATGATAATGAATGTCCTATAGTACATAGATCTGCTTATG
ATGCACTTCCCTCAACAACTATTGTATCAATGGCTTGTTGTGCCTC
AGGTTCTACTAGAGGTTATGATGAATTGGTCCCTCATCAAATATCT
GTGGTATCAGAAGAAAGATTTTACACAAAATGGAATCCCGAGGCT
CTCCCAAGCAATACTGGAGAAGTTAATTTTCAAAGTGGAATTATA
GCAGCTAGGTGTGCTATAAGTAAATTGCATCAAGAACTTGGTGCA
AAAGGATTTATTCAAGTTTATGTAGATCAAGTAGATGAAGATATT
GTAGCAGTTACTAGACATAGTCCTAGTATACATCAAAGTGTTGTAG
CAGTATCCAGAACTGCTTTTAGAAATCCTAAAACTAGCTTTTATAG
TAAAGAAGTTCCTCAAATGTGTATTCCTGGAAAAATTGAAGAAGT
TGTATTGGAAGCAAGAACTATAGAAAGGAATACTAAACCCTATAG
AAAAGATGAAAATTCTATAAATGGTACTCCTGATATTACTGTGGA
AATAAGAGAACATATACAACTTAATGAAAGCAAAATTGTAAAACA
AGCTGGTGTTGCTACAAAAGGTCCTAATGAATATATCCAAGAAAT
TGAATTTGAAAACCTCTCCCCTGGTTCTGTAATTATATTTAGAGTA
TCATTGGACCCTCATGCACAAGTTGCTGTTGGTATTCTCAGAAATC
ATTTGACACAATTTTCTCCTCATTTTAAATCTGGATCATTGGCTGTA
GATAATGCAGATCCTATACTTAAAATTCCCTTTGCATCATTAGCTA
GTAGACTTACCTTGGCAGAACTGAATCAAATACTCTATAGGTGTG
AATCTGAAGAAAAAGAAGATGGTGGAGGTTGTTATGATATTCCTA
ATTGGTCTGCTTTGAAATATGCAGGTTTGCAAGGTTTAATGTCTGT
TCTTGCAGAAATAAGACCAAAAAATGATTTGGGTCATCCATTTTGT
AATAATCTGAGAAGTGGTGATTGGATGATAGATTATGTAAGTAAT
AGATTGATTAGTAGAAGTGGTACAATAGCTGAAGTTGGTAAATGG
TTGCAAGCTATGTTTTTTTACCTCAAACAAATCCCAAGATACCTTA
TTCCATGTTATTTTGATGCAATTCTTATAGGAGCATATACTACTTTA
TTGGATACAGCATGGAAACAAATGICAAGITTTGTACAAAATGGT
TCAACTTTTGTAAAACACCTTTCACTTGGAAGTGTTCAATTATGTG
GTGTAGGGAAATTTCCTTCCTTGCCTATTCTGTCACCTGCTTTGATG
GATGTACCATATAGATTGAATGAAATAACCAAAGAAAAAGAACA
ATGTTGTGTTAGCTTGGCAGCAGGTTTACCTCATTTTAGTTCAGGA
ATTTTTAGATGTTGGGGTAGAGATACATTTATAGCTCTTAGAGGAA
TTTTGTTGATAACAGGAAGATATGTTGAAGCAAGAAATATAATAT
TGGCATTTGCAGGTACACTTAGACATGGTTTGATTCCAAATCTTTT
GGGTGAAGGTATTTATGCTAGATATAATTGTAGAGATGCTGTTTGG
TGGTGGTTACAATGTATACAAGATTACTGTAAAATGGTACCTAATG
GACTTGATATATTGAAGTGTCCAGTTTCAAGAATGTATCCTACAGA
TGATTCTGCACCACTCCCTGCTGGTACTTTGGATCAACCTCTGTTTG
AAGTTATACAGGAAGCTATGCAGAAACATATGCAAGGTATTCAAT
TTAGAGAAAGAAATGCAGGTCCTCAAATTGATAGGAATATGAAAG

Name Sequence ATGAAGGATTTAACATAACTGCTGGAGTAGATGAAGAAACTGGAT
TTGTCTATGGTGGAAACAGATTTAATTGTGGTACATGGATGGATAA
AATGGGTGAATCTGATAGAGCTAGAAATAGAGGTATTCCAGCAAC
ACCAAGAGATGGTTCTGCAGTAGAAATTGTAGGTTTGAGTAAATC
AGCTGTTAGATGGCTCTTGGAACTCTCTAAAAAAAATATATTTCCT
TATCATGAGGTAACCGTAAAAAGACATGGAAAAGCTATTAAAGTT
TCTTATGATGAATGGAATAGAAAAATTCAAGATAATTTTGAGAAA
CTTTTTCATGTGTCTGAAGACCCATCTGATTTGAATGAAAAGCATC
CCAATCTTGTCCATAAAAGAGGAATTTATAAAGATAGTTATGGAG
CATCATCTCCTTGGTGTGATTATCAATTGAGACCAAATTTTACTAT
TGCTATGGTTGTAGCTCCTGAGTTGTTTACAACAGAAAAGGCTTGG
AAAGCCTTGGAAATTGCAGAAAAAAAACTCCTTGGTCCACTGGGT
ATGAAAACACTTGATCCTGATGATATGGTATATTGTGGTATTTATG
ATAATGCATTGGATAATGATAACTACAATCTTGCTAAAGGATTTAA
TTACCATCAAGGACCTGAATGGTTGTGGCCAATTGGTTATTTTTTG
AGAGCAAAACTTTATTTTTCTAGGTTGATGGGACCAGAAACTACA
GCTAAAACAATTGTTTTGGTGAAGAATGTTCTTTCAAGACATTATG
TACATTTGGAAAGATCACCTTGGAAAGGTCTTCCAGAACTTACTAA
TGAAAATGCACAATATTGTCCATTTTCCTGTGAAACTCAAGCATGG
TCCATAGCCACTATATTGGAGACCCTTTATGACTTGTA (SEQ ID NO:
179) 1002191 In one aspect, a codon optimized, engineered nucleic acid sequence encoding human GDE is provided. In certain embodiments, an engineered human GDE cDNA is provided herein (as SEQ ID NO: 175), which was designed to maximize translation as compared to the native GDE sequence (SEQ ID NO: 174). Preferably, the codon optimized GDE coding sequence has less than about 80% identity, preferably about 75%
identity or less to the full-length native GDE coding sequence (SEQ ID NO: 174). In one embodiment, the codon optimized GDE coding sequence has about 75% identity with the native GDE
coding sequence of SEQ ID NO: 174. In one embodiment, the codon optimized GDE coding sequence is characterized by improved translation rate as compared to native GDE following delivery. In one embodiment, the codon optimized GDE coding sequence shares less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity to the full length native GDE
coding sequence of SEQ ID NO: 174. In one embodiment, the codon optimized nucleic acid sequence is a variant of SEQ TD NO: 175. Tn another embodiment, the codon optimized nucleic acid sequence a sequence sharing about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%

or greater identity with SEQ ID NO: 175. In one embodiment, the codon optimized nucleic acid sequence is SEQ ID NO: 175. In another embodiment, the nucleic acid sequence is codon optimized for expression in humans. In other embodiments, a different GDE coding sequence is selected.
1002201 In one aspect, a CpG minimized, engineered nucleic acid sequence encoding human GDE is provided. In certain embodiments, an engineered human GDE cDNA is provided herein (as SEQ ID NO: 179), which was designed to minimize CpG motifs as compared to the native GDE sequence (SEQ ID NO: 174). Preferably, the CpG
minimized GDE coding sequence has less than about 90% identity, preferably about 85%
identity or less to the full-length native GDE coding sequence (SEQ ID NO: 174). In one embodiment, the CpG minimized GDE coding sequence has about 81% identity with the native GDE
coding sequence of SEQ ID NO: 174. In one embodiment, the CpG minimized GDE coding sequence is characterized by a reduced activation for host immune reaction as compared to native GDE sequence following delivery into host cells. In one embodiment, the CpG
minimized GDE coding sequence shares less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity to the full length native GDE coding sequence of SEQ ID
NO: 174. In one embodiment, the CpG minimized nucleic acid sequence is a variant of SEQ ID
NO: 179.
In another embodiment, the CpG minimized nucleic acid sequence has a sequence sharing about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or greater identity with SEQ ID NO:
179. In one embodiment, the CpG minimized nucleic acid sequence is SEQ ID NO: 179.
1002211 In some embodiments, a hairpin-ended DNA molecule, as described herein, encodes a fusion protein comprising a full length, fragment or portion of a GDE protein fused to another sequence (e.g. , an N or C terminal fusion). In some embodiments, the N or C
terminal sequence is a signal sequence or a cellular targeting sequence.
1002221 In a specific embodiment, an expression cassette comprises a GDE
transgene that is at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence set forth in SEQ ID NO: 174. In a specific embodiment, an expression cassette comprises a GDE
transgene that is at least 60%, at least 70%, at least 80% or at least 90%
identical to the sequence set forth in SEQ ID NO: 175. In a specific embodiment, an expression cassette comprises a GDE transgene that is at least 60%, at least 70%, at least 80% or at least 90%

identical to the sequence set forth in SEQ ID NO: 179. In a specific embodiment, an expression cassette comprises a GDE transgene that is at least 60%, at least 70%, at least 80%
or at least 90% identical to the sequence set forth in SEQ ID NO: 178. In a specific embodiment, an expression cassette comprises a GDE transgene that is at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence set forth in SEQ
ID NO: 179.
[00223] In a specific embodiment, an expression cassette comprises a GDE
transgene that is identical to the sequence set forth in SEQ ID NO: 174. In a specific embodiment, an expression cassette comprises a GDE transgene that is identical to the sequence set forth in SEQ ID NO: 175. In a specific embodiment, an expression cassette comprises a GDE
transgene that is identical to the sequence set forth in SEQ ID NO: 179. In a specific embodiment, an expression cassette comprises a GDE transgene that is identical to the sequence set forth in SEQ ID NO: 178. In a specific embodiment, an expression cassette comprises a GDE transgene that is identical to the sequence set forth in SEQ
ID NO: 179 [00224] The term "percent (%) identity", "sequence identity", "percent sequence identity", or "percent identical" in the context of GDE endcoding nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
1002251 Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences.
A suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids. Generally, when referring to "identity", "homology", or "similarity"
between two different sequences, "identity", "homology" or "similarity" is determined in reference to "aligned" sequences. "Aligned" sequences or "alignments" refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
1002261 Identity may be determined by preparing an alignment of the sequences and through the use of a variety of algorithms and/or computer programs known in the art or commercially available [e.g., BLAST, ExPASy; Clustal0; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith-Waterman algorithm]. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs.
Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal Omega", and "Clustal X", programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed.
Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs.
See, e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999). Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible through Web Servers on the interne.
1002271 Codon-optimized coding regions can be designed by various different methods.
This optimization may be performed using methods which are available on-line (e g , GeneArt), published methods, or a company which provides codon optimizing services, e.g., DNA2.0 (Menlo Park, CA). Suitably, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide. A number of options are available for performing the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological manipulations well known to those of ordinary skill in the art.
1002281 The GDE expression cassette may be located at any suitable distance of base pairs from either the 5' and/or 3' ITR closing pair (as described in section 5.4.1) to allow or to maintain efficient transcription of said expression cassette in host cells. In some embodiments the distance between the expression cassette and the 5' ITR and the distance between the expression cassette and the 3' ITR closing pair are identical. In some embodiments the distance between the expression cassette and the 5' ITR and the distance between the expression cassette and the 3' ITR closing pair are not identical. In some embodiments the distance between the expression cassette and/or the 3' ITR closing pair and the distance between the expression cassette the 5' ITR closing pair is least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, or at least 400 nucleotides. In some embodiments the distance between the expression cassette and the 3' ITR closing pair and/or the distance between the expression cassette and the 5' ITR closing pair is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, or about 400 nucleotides.
1002291 By "engineered nucleic acid sequence" is meant that the nucleic acid sequences encoding the GDE protein described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the GDE sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like) or for generating viral vectors in a packaging host cell and/or for delivery to a host cells in a subject. In one embodiment, the genetic element is a circular plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
1002301 In one embodiment, the nucleic acid sequence encoding GDE further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. The tag polypeptide may be selected from known "epitope tags" including, without limitation, a myc tag polypeptide, a glutathione-S-transferase tag polypeptide, luciferase protein tag polypeptide, a green fluorescent protein tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His6 tag polypeptide, an influenza virus hemagglutinin tag polypeptide, a flag tag polypeptide, and a maltose binding protein tag polypeptide. In some aspects, hairpin ended vectors expressing an GDE protein linked to a reporter polypeptide may be used for diagnostic purposes, as well as to determine efficacy or as markers of the hairpin ended vectors' activity in the subject to which they are administered.
5.4.4 Hairpin-ended DNA molecules encoding GDE
1002311 As is clear from the description above, the hairpin-ended DNA
molecules for expressing a human amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase provided herein comprise an expression cassette An "expression cassette" is a nucleic acid molecule or a part of nucleic acid molecule containing sequences or other information that directs the cellular machinery to make RNA and protein. An expression cassette can comprise a transcription unit or an open reading frame (ORF) encoding the GDE protein or fragment thereof. In some embodiments, an expression cassette comprises a promoter sequence. In yet some other embodiments, an expression cassette comprises a promoter operatively linked to the transcription unit. The expression cassette can further comprise features to direct the cellular machinery to make RNA and protein. In one embodiment, the expression cassette comprises a posttranscriptional regulatory element. In another embodiment, the expression cassette further comprises a polyadenylation and/or termination signal. In yet another embodiment, the expression cassette comprises regulatory elements known and used in the art to regulate (promote, inhibit and/or turn on/off the expression of the ORF). Such regulatory elements include, for example, 5'-untranslated region (UTR), 3'-UTR, or both the 5'UTR and the 3'UTR In some further embodiments, the expression cassette comprises any one or more features provided in this Section (Section 5.4.3) in any combination or permutation.
1002321 The expression cassette can comprise a protein coding sequence in its ORF (sense strand). Alternatively, the expression cassette can comprise the complementary sequence of the protein coding ORF (anti-sense strand) and the regulatory components and/or other signals for the cellular machinery to produce a sense strand DNA/RNA and the corresponding protein. In some embodiments, the expression cassette comprises a GDE
protein sequence without intron. In other embodiments, the expression cassette comprises a GDE protein sequence with intron, which is removed upon transcription and splicing. The expression cassette can also comprise various numbers of ORFs or transcription units. In one embodiment, the expression cassette comprises 1, 2, 3, 4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ORFs. In another embodiment, the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transcription units.
1002331 The expression cassettes can also comprise one or more transcriptional regulatory element, one or more posttranscriptional regulatory elements, or both one or more transcriptional regulatory element and one or more posttranscriptional regulatory elements.
Such regulatory elements are any sequences that allow, contribute or modulate the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (e.g.
mRNA) into the host cell or organism. Such regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc.
1002341 In some embodiments, the expression cassette comprises an enhancer.
Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. In some embodiments, an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. In certain specific embodiments, the enhance can be Woodchuck HBV
Post-transcriptional regulatory element (WPRE), intron/exon sequence derived from human apolipoprotein Al precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit13-globin intron, a P5 promoter of an AAV, or any combination thereof.
1002351 As described above, the expression cassette can comprise a promoter to control expression of a protein of interest. Promoters include any nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. Promoters can be a constitutive, inducible, or repressible. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (e.g., derived from the same genetic source) or a heterologous promoter (e.g., derived from a different genetic source). In some embodiments, a promoters can be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. In other embodiments, a promoter can be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. In further embodiments, a promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic to promote expression in cells or tissues in which expression of GDE is desirable such as in cells or tissues in which GDE
expression is desirable in GDE-deficient patients.
1002361 ln a particular embodiment, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include the muscle creatine kinase (MCK) promoter. Non-limiting examples of suitable muscle creatine kinase promoters are human muscle creatine kinase promoters and truncated murine muscle creatine kinase [(tMCK) promoters] (Wang B et al, Construction and analysis of compact muscle-selective promoters for AAV vectors. Gene Ther. 2008 Nov;15(22): 1489-99) (representative GenBank Accession No. AF188002). Human muscle creatine kinase has the Gene 1D No. 1158 (representative GenBank Accession No. NC 000019.9). Other examples of muscle-specific promoters include a synthetic promoter C5.12 (spC5. 12, alternatively referred to herein as "C5.12-), such as the spC5.12 or the spC5. 12 promoter (disclosed in Wang et al., Gene Therapy volume 15, pages 1489-1499 (2008)), the WICK7 promoter (Salva et al. Mol Ther. 2007 Feb;15(2):320-9), myosin light chain (MLC) promoters, for example MLC2 (Gene 1D No.
4633; representative GenBank Accession No. NG 007554.1); myosin heavy chain (MHC) promoters, for example alpha-WIC (Gene 1D No. 4624; representative GenBank Accession No. NG 023444.1); desmin promoters (Gene 1D No. 1674; representative GenBank Accession No. NG 008043.1); cardiac troponin C promoters (Gene 1D No. 7134;
representative GenBank Accession No. NG 008963.1); troponin I promoters (Gene ID Nos.
7135, 7136, and 7137; representative GenBank Accession Nos. NG 016649.1, NG
011621.1, and NG 007866.2,); myoD gene family promoters (Weintraub et al., Science, 251 , 761 (1991); Gene ID No. 4654; representative GenBank Accession No. NM 002478);
alpha actin promoters (Gene ID Nos. 58, 59, and 70; representative GenBank Accession Nos.
NG
006672.1, NG 011541.1, and NG 007553.1,); beta actin promoters (Gene ID No.
60;
representative GenBank Accession No. NG 007992.1); gamma actin promoters (Gene ID No.
71 and 72; representative GenBank Accession No. NG 011433.1 and NM 001199893);

muscle-specific promoters residing within intron 1 of the ocular form of Pitx3 (Gene ID No.
5309) (Coulon et al; the muscle-selective promoter corresponds to residues 11219-11527 of representative GenBank Accession No. NG 008147); and the promoters described in US

Patent Publication US 2003/0157064, and CK6 promoters (Wang et al 2008 doi:
10.1038/gt.2008.104). In another particular embodiment, the muscle-specific promoter is the E-Syn promoter described in Wang et al., Gene Therapy volume 15, pages 1489-1499 (2008), comprising the combination of a MCK- derived enhancer and of the spC5.12 promoter. In a particular embodiment of the disclosure, the muscle- specific promoter is selected in the group consisting of a spC5.12 promoter, the MHCK7 promoter, the E-syn promoter, a muscle creatine kinase myosin light chain (MLC) promoter, a myosin heavy chain (MHC) promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an alpha actin promoter, an beta actin promoter, an gamma actin promoter, a muscle-specific promoter residing within intron 1 of the ocular form of Pitx3, a CK6 promoter, a CK8 promoter and an Actal promoter. In a particular embodiment, the muscle-specific promoter is selected in the group consisting of the spC5.12, desmin and MCK promoters. In a further embodiment, the muscle-specific promoter is selected in the group consisting of the spC5.12 and MCK
promoters. In a particular embodiment, the muscle-specific promoter is the spC5.12 promoter.
1002371 In a particular embodiment, the promoter is a liver-specific promoter.
Non-limiting examples of liver- specific promoters include the alpha- 1 antitrypsin promoter (hAAT), the transthyretin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the LSP promoter (comprising a thyroid hormone-binding globulin promoter sequence, two copies of an alpha-microglobulin/bikunin enhancer sequence, and a leader sequence - Ill, C. R., et al. (1997). Optimization of the human factor VIII
complementary DNA expression plasmid for gene therapy of hemophilia A. Blood Coag.
Fibrinol. 8: S23-S30), etc. Other useful liver-specific promoters are known in the art, for example those listed in the Liver Specific Gene Promoter Database compiled the Cold Spring Harbor Laboratory (http://rulai.cshl.edu/LSPD/). A preferred liver-specific promoter in the context of the disclosure is the hAAT promoter. In another particular embodiment, the promoter is a neuron-specific promoter. Non-limiting examples of neuron-specific promoters include, but are not limited to the following: synapsin-1 (Syn) promoter, neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad.
Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al.
Neuron, 15:373-84 (1995)), among others which will be apparent to the skilled artisan. In a particular embodiment, the neuron-specific promoter is the Syn promoter. Other neuron-specific promoters include, without limitation: synapsin-2 promoter, tyrosine hydroxylase promoter, dopamine b-hydroxylase promoter, hypoxanthine phosphoribosyltransferase promoter, low affinity NGF receptor promoter, and choline acetyl transferase promoter (Bejanin et al., 1992;
Carroll et al., 1995; Chin and Greengard, 1994; Foss-Petter et al., 1990;
Harrington et al., 1987; Mercer et al., 1991; Patei et al., 1986). Representative promoters specific for the motor neurons include, without limitation, the promoter of the Calcitonin Gene-Related Peptide (CGRP), a known motor neuron- derived factor. Other promoters functional in motor neurons include the promoters of Choline Acetyl Transferase (ChAT), Neuron Specific Enolase (NSE), Synapsin and Hb9. Other neuron-specific promoters useful in the present disclosure include, without limitation: GFAP (for astrocytes), Calbindin 2 (for intemeurons), Mnxl (motomeurons), Nestin (neurons), Parvalbumin, Somatostation and Plpl (oligodendrocytes and Schwann cells). In another particular embodiment, the promoter is a ubiquitous promoter.
Representative ubiquitous promoters include the cytomegalovirus enhancer/chicken beta actin (CAG) promoter, the cytomegalovirus enhancer/promoter (CMV) (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the PGK
promoter, the SV40 early promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 alpha promoter. ln addition, the promoter may also be an endogenous promoter such as the albumin promoter or the GDE
promoter. ln a particular embodiment, the promoter is associated to an enhancer sequence, such as a cis-regulatory module (CRMs) or an artificial enhancer sequence.
CRMs useful in the practice of the present disclosure include those described in Rincon et al., Mol Ther. 2015 Jan,23(1):43-52, Chuah et al., Mol Ther. 2014 Sep;22(9): 1605-13 or Nair et al., Blood. 2014 May 15; 123(20):3195-9. Other regulatory elements that are, in particular, able to enhance muscle-specific expression of genes, in particular expression in cardiac muscle and/or skeletal muscle, are those disclosed in W02015110449. Particular examples of nucleic acid regulatory elements that comprise an artificial sequence include the regulatory elements that are obtained by rearranging the transcription factor binding sites (TFBS) that are present in the sequences disclosed in W02015110449. Said rearrangement may encompass changing the order of the TFBSs and/or changing the position of one or more TFBSs relative to the other TFBSs and/or changing the copy number of one or more of the TFBSs. For example, a nucleic acid regulatory element for enhancing muscle-specific gene expression, in particular cardiac and skeletal muscle-specific gene expression, may comprise binding sites for E2A, HNH 1, NF1, C/EBP, LRF, MyoD, and SREBP; or for E2A, NF1, p53, C/EBP, LRF, and SREBP; or for E2A, HNH 1, HNF3a, HNF3b, NF1, C/EBP, LRF, MyoD, and SREBP, or E2A, HNF3a, NF I, C/EBP, LRF, MyoD, and SREBP; or for E2A, HNF3a, NF'1, CEBP, LRF, MyoD, and SREBP; or for HNF4, NF I, RSRFC4, C/EBP, LRF, and MyoD, or NFI
, PPAR, p53, C/EBP, LRF, and MyoD. For example, a nucleic acid regulatory element for enhancing muscle-specific gene expression, in particular skeletal muscle-specific gene expression, may also comprise binding sites for E2A, NF I, SRFC, p53, C/EBP, LRF, and MyoD; or for E2A, NF1, C/EBP, LRF, MyoD, and SREBP; or for E2A, HNF3a, C/EBP, LRF, MyoD, SEREBP, and Tall b; or for E2A, SRF, p53, C/EBP, LRF, MyoD, and SREBP;
or for HNF4, NF I, RSRFC4, C/EBP, LRF, and SREBP; or for E2A, HNF3a, HNF3b, NF1, SRF, C/EBP, LRF, MyoD, and SREBP; or for E2A, CEBP, and MyoD. In further examples, these nucleic acid regulatory elements comprise at least two, such as 2, 3, 4, or more copies of one or more of the TFBSs recited before. Other regulatory elements that are, in particular, able to enhance liver-specific expression of genes, are those disclosed in W02009130208.
Table 19: Exemplary Regulatory Elements Description Sequence Endogenous AGTTGCGGAGCGATCCTTTTAAAAGGTCAATCAGATTATGTCA
hAGL CTCCTCTGTTCAAAATCTCCATGCCTTTTTTTCCAAAGTTTAAA
Promoter AGCCCAAGTCCTTGCATTGGCCTACAAAGCCTTAAAAGATCTG
GTCACCCGTCTGTGCTCCTGATCCCTTCTCCTGCCCCATCTGTC
TGTCCTAATCTCTTACCACTCTCTTCTTCACTCAGCTATGGTGA
TCTTCTTCGGTTCACCAAGTATGCTCCTGCCTCATGGCCTTTGT
ACTTCCTATACCICTACATGTAACCCTCTACCTTAGACTICTTC
TTTCTCGCAGTTTGGCATCACTTTACTGAACGTTATATTTAGAA
ATGCAAACCTCTCTCTGCTTACTCTTCCACACTTCCCCCTTCCT
ATTATATATAGGATATAACATATCCTTCTTATTATATAGGATAT
ATTATATCCTGTATAATTTATTTAATTTATCTGTCTCCCACCAA
TAAAATGTAGGAGTTCCTTGAGGGCAGTGACTGTTTTATTGCT
GCATTCCCAGCACCTTATGTGCCTGGCAAATAGTAGGGGCCAG
AAAATGAGCTGTGGGTTCCCAAAGTCAGTTACGGACCATTTGC
AACTAGCCATTCTCAGAAATCTACAGAAATAAACAAATACTTC
AGTATGGGGTTTTTTTTTTTAACTTATATCCTCTTTGGACCTAC
AGTCATTCCACAATAAAGAATGCAAGAATCTTCTCCACACGCC
ACAAGTCTTAGTTAACCAAATCTTCTGTCCATTTTCTCATAACC
ATTAGGAGCCCTCCAAAAGCCCTGGAAGATGGGTTTTCCTTTA
CCCTCAGGCATTAAATCTCCTTAAGCATCTGCAAAAAGTTCTG
AGTTACTGGCCTAACATAAGTGCAGCTTAATCTCAGACGATCT
CCGGGTCTATCTAGTGTACATGAGGTACACCCGGACACCGTTA
AGTATCAGTGGTGTTTGCACTCTCGATGGTTTGCAGACTGGCC
ACACCTTACCTACTGGGTCTGCATTCAGGAACATGTGTCCTGT
CTGTTAGCACTAGAAGTGATGGACACGTGTTGGCTGGAATGTC
AAGGCTGTAGCCAGGCCCCTTATTTTAGACACTTAGAAATCAG
GACTCTGAGAACTTAGGCCAAGTAAAAATTATCAAAACAAAG
AAACAAAACACGTGGTGGCACAAAAGACACCAGAAGCCAGGT

Description Sequence CGTTTGCCCCTCACCATTCAGCCCTTCCCAGCAAAAGATCCTA
CTGTGCAGCTCAACCTAGCTCGCAGCCGGTACCGCGGGATTTT
AATGTGCAACTGTGAGCTCGCAGGCTGTTAAAGGAAGGCCGC
GCCTTGGCCGGTGCACCTTCCCCAGGGCAAGGAGAAAGCGCC
GCTCCCGGCCTCAGCCGCAGCAGGCTCCAGGTCCCCCGGCCCG
GAGCCGACTGAGACGGTGCGGTGCCCACGCTCTCGCGAGACT
AGCGGTCGGGGCGGGC GGGTC GAGC C T C CC GGAAGT GGGC C A
GAGGTACGGTCCGCTCCCACCTGGGGCGAGTGCGCGCACGGC
CAGGTTGGGTACCGGGTGCGCCCAGGAACCCGCGCGAGGCGA
AGTC GCTGAGACTC TGCC TGC TTCT CAC CCAGC TGCCTCGGCG
CTGCCCCGGTCGCTCGCCGCCCCTCCCTTTGCCCTTCACGGCGC
CCGGCCCTCCTTGGGCTGCGGCTTCTGTGCGAGGCTGGGCAGC
CAGCCCTTCCCCTTCTGTTTCTCCCCGTCCCCTCCCCCCGACCG
TAGC (SEQ ID NO: 181) Endogenous CCTGGAAGATGGGTTTTCCTTTACCCTCAGGCATTAAATCTCCT
hAGL TAAGCATCTGCAAAAAGTTCTGAGTTACTGGCCTAACATAAGT
promoter (agl) GCAGCTTAATCTCAGACGATCTCCGGGTCTATCTAGTGTACAT
GAGGTAC AC C C GGAC AC C GTTAAGTAT C AGT GGTGT TT GC AC T
CTCGATGGTTTGCAGACTGGCCACACCTTACCTACTGGGTCTG
CATTCAGGAACATGTGTCCTGTCTGTTAGCACTAGAAGTGATG
GACACGTGTTGGCTGGAATGTCAAGGCTGTAGCCAGGCCCCTT
ATTTTAGACACTTAGAAATCAGGACTCTGAGAACTTAGGCCAA
GTAAAAATTATCAAAACAAAGAAACAAAACACGTGGTGGCAC
AAAAGACACCAGAAGCCAGGTCGTTTGCCCCTCACCATTCAGC
CCTTCCCAGCAAAAGATCCTACTGTGCAGCTCAACCTAGCTCG
CAGCCGGTACCGCGGGATTTTAATGTGCAACTGTGAGCTCGCA
GGCTGTTAAAGGAAGGCCGCGCCTTGGCCGGTGCACCTTCCCC
AGGGCAAGGAGAAAGCGCCGCTCCCGGCCTCAGCCGCAGCAG
GCTCCAGGTCCCCCGGCCCGGAGCCGACTGAGACGGTGCGGT
GCCCACGCTCTCGCGAGACTAGCGGTCGGGGCGGGCGGGTCG
AGCC TC C C GGAAGTGGGC CAGAGGTAC GGTC C GC TC C C AC C TG
GGGCGAGTGCGCGCACGGCCAGGTTGGGTACCGGGTGCGCCC
AGGAAC C C GC GC GAGGC GAAGTC GC T GAGAC TC TGC C T GC T TC
TCACCCAGCTGCCTCGGCGCTGCCCCGGTCGCTCGCCGCCCCT
CCCTTTGCCCTTCACGGCGCCCGGCCCTCCTTGGGCTGCGGCTT
CTGTGCGAGGCTGGGCAGCCAGCCCTTCCCCTTCTGTTTCTCCC
CGTCCCCTCCCCCCGACCGTAGC (SEQ ID NO: 183) Endogenous AATC AC TAC TAAAGGAATTGATGTCATCAATATC TT TTAC TC C T
hAGL TATAT C TAATT GCAAC AC T GGGCAT TAAAGTGAGAGTT TTAC T
Enhancer GGAGGAAGGACAGCAAGAAAGGCTAATTTTGGAGCCCTGGAG
AACAGTGATCAACAGGAGGGCAGTGTAATGAGATAGTCATAG
GAGAGACTGAAAGTGGGAGGGGGCATGGAAAGGGAGAACTT
GAAGACAAACATAAATGTGATCTGTTTTCACAACATGGTCAGG
GC C TC AC TC TGC TAAC ATTTGTATGTAC GC TAGTAC TTAGTC TC
TATCAGGCACAGTICIAAGCCCICATITACTIAACAATAGATA
CTACTTTCATCCCCATTTTATAGTTGCAAAAACCAAGGCCCAA
AGAGGTTGAGTACCAT (SEQ ID NO: 184) ApoE AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCC
enhancer- TTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCT

Description Sequence hAAT GCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTC
promoter- CCTAAAATGGGCAAACTTTGCAAGCAGCAAACAGCAAACACA
SpC5.12 CAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCA
promoter GAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACC
CCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTG
GTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACAGC
CACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGG
TACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTT
GGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCC
TTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGC
GTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGG
ACTTAGCCCCTGITTGCTCCTCCGATAACTGGGGTGACCTIGGT
TAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGtaCCACT
GCTTAAATACGGACGAGGACAGGTCTAGATGGCCACCGCCTTC
GGCACCATCCTCACGACACCCAAATATGGCGACGGGTGAGGA
ATGGTGGGGAGTTATTTTTAGAGCGGTGAGGAAGGTGGGCAG
GCAGCAGGTGTTGGCGCTCTAAAAATAACTCCCGGGAGTTATT
TTTAGAGCGGAGGAATGGTGGACACCCAAATATGGCGACGGT
TCCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGG
CCGCATTCCTGGGGGCCGGGCGGTGCTCCCGCCCGCCTCGATA
AAAGGCTCCGGGGCCGGCGGCGGCCCACGAGCTACCCGGAGG
AGCGGGAGGCGCCAAGCTCTAGATCTAGAAAGAGGTAAGGGT
TTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCT
GGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG (SEQ ID
NO: 185) 1002381 In some embodiments, the expression cassette can comprise a polyadenylation, termination signal, or both a polyadenylation and termination signal. Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. In some embodiments, the polyadenylation signal can be a SV40 polyadenylation signal, polyadenylation signal (bp 4411-4466, NC 001401), a polyadenylation signal from the Herpes Simplex Virus Thymidine Kinase Gene, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human P-globin polyadenylation signal.
1002391 In some embodiments the expression cassette can have various sizes to accommodate one or more ORFs of various lengths. In certain embodiments, the size of expression cassette at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, or at least 80 kb. In one specific embodiment, the expression cassette is at least 4.5 kb.

In another specific embodiment, the expression cassette is at least 4.6 kb. In yet another specific embodiment, the expression cassette is at least 4.7 kb. In a further specific embodiment, the expression cassette is at least 4.8 kb. In one specific embodiment, the expression cassette is at least 4.9 kb. about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, about 10 kb, about 15 kb, about 20 kb, about 25 kb, about 30 kb, about 35 kb, about 40 kb, about 45 kb, about 50 kb, about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, or about 80 kb. In one specific embodiment, the expression cassette is about 4.5 kb. In another specific embodiment, the expression cassette is about 4.6 kb. In yet another specific embodiment, the expression cassette is about 4.7 kb. In a further specific embodiment, the expression cassette is about 4.8 kb. In one specific embodiment, the expression cassette is about 4.9 kb. In another specific embodiment, the expression cassette is about 5 kb. The expression cassette can also comprise various numbers of genes of interest ("transgenes"). In one embodiment, the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transgenes. In some specific embodiment, the expression cassette comprise one transgene. In some embodiments, the transgenes are recombinant genes. In some further embodiments, the transgenes comprise cDNA sequences (e.g no introns in the transgenes).
1002401 In some embodiment, the DNA molecules provided herein do not have the size limitations of encapsidated AAV vectors, thus enabling delivery of a large-size expression cassette to provide efficient transgene. In certain embodiments, the DNA
molecules provided herein comprise expression cassette equal to or larger than the size of any natural AAV
genome.
1002411 The expression cassette can have various positions relative to the inverted repeat.
In some embodiments, the expression cassette is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides apart from the inverted repeat. In certain embodiments, the expression cassette is at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6, at least kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 1.5kb, or at least 2 kb apart from the inverted repeat. In other embodiments, the expression cassette is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides apart from the inverted repeat. In further embodiments, the expression cassette is about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6, about kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.5kb, or about 2 kb apart from the inverted repeat. In one embodiment, the inverted repeat in this paragraph is the first inverted repeat as described in Sections 3 and 5.4 (including 5.4.1). In another embodiment, the inverted repeat in this paragraph is the second inverted repeat as described in Sections 3 and 5.4 (including 5.4.1) In yet another embodiment, the inverted repeat in this paragraph is both the first and the second inverted repeat as described in Sections 3 and 5.4 (including 5.4.1) 1002421 In one aspect, provided herein is a double-stranded DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a sense strand 5' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE protein;
and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a sense strand 3' overhang comprising the second inverted repeat upon separation of the top from the antisense strand of the second inverted repeat (e.g.
as described in Sections 5.3.3, 5.3.4 and 5.4.2) 1002431 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in an antisense strand 3' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE
protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in an antisense strand 5' overhang comprising the second inverted repeat upon separation of the sense from the antisense of the second inverted repeat 1002441 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a sense strand 5' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE
protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in an anti sense strand 5' overhang comprising the second inverted repeat upon separation of the sense from the antisense strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2).
1002451 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in an antisense strand 3' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE
protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a sense strand 3' overhang comprising the second inverted repeat upon separation of the sense from the antisense strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2 or depicted in FIGS. 2B
and 2C).
1002461 In one aspect, provided herein is a double-stranded DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a sense strand 5' overhang comprising the first inverted repeat upon separation of the sense from the anti sense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a sense strand 3' overhang comprising the second inverted repeat upon separation of the sense from the antisense of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2).
1002471 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in an anti sense strand 3' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in an antisense strand 5' overhang comprising the second inverted repeat upon separation of the sense from the antisense strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2).
1002481 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a sense strand 5' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE protein;
and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in an antisense strand 5' overhang comprising the second inverted repeat upon separation of the sense from the antisense strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2).
1002491 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the sense strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in an antisense strand 3' overhang comprising the first inverted repeat upon separation of the sense from the antisense strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) a sense expression cassette encoding a therapeutic GDE protein; and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third and a fourth target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a sense strand 3' overhang comprising the second inverted repeat upon separation of the sense from the anti sense strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2 or depicted in FIGS. 2B and 2C). In one embodiment, the first, second, third, and fourth target site for programmable nicking enzyme in this and the preceding three paragraphs are all the same. In another embodiment, three of the first, second, third, and fourth target site for programmable nicking enzyme in this and the preceding three paragraphs are the same. In yet another embodiment, two of the first, second, third, and fourth target site for programmable nicking enzyme in this and the preceding three paragraphs are the same. In a further embodiment, the first, second, third, and fourth target site for programmable nicking enzyme in this and the preceding three paragraphs are all different.
1002501 The expression cassettes can also comprise one or more transcriptional regulatory element, one or more posttranscriptional regulatory elements, or both one or more transcriptional regulatory element and one or more posttranscriptional regulatory elements.
Such regulatory elements are any sequences that allow, contribute or modulate the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (e.g.
mRNA) into the host cell or organism. Such regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc.
1002511 The expression cassette can have various sizes to accommodate one or more ORFs of various lengths. In certain embodiments, the size of expression cassette is at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6, at least kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 1.5kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, or at least 80 kb. In one specific embodiment, the expression cassette is at least 4.5 kb. In another specific embodiment, the expression cassette is at least 4.6 kb. In yet another specific embodiment, the expression cassette is at least 4.7 kb. In a further specific embodiment, the expression cassette is at least 4.8 kb. In one specific embodiment, the expression cassette is at least 4.9 kb. In another specific embodiment, the expression cassette is at least 5 kb. In other embodiments, the size of the expression cassette is about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6, about kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.5kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, about 10 kb, about 15 kb, about 20 kb, about 25 kb, about 30 kb, about 35 kb, about 40 kb, about 45 kb, about 50 kb, about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, or about 80 kb. In one specific embodiment, the expression cassette is about 4.5 kb. In another specific embodiment, the expression cassette is about 4.6 kb. In yet another specific embodiment, the expression cassette is about 4.7 kb. In a further specific embodiment, the expression cassette is about 4.8 kb. In one specific embodiment, the expression cassette is about 4.9 kb. In another specific embodiment, the expression cassette is about 5 kb. The expression cassette can also comprise various numbers of genes of interest ("transgenes"). In one embodiment, the expression cassette comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transgenes. In some specific embodiment, the expression cassette comprise one transgene.
In some embodiments, the transgenes are recombinant genes. In some further embodiments, the transgenes comprise cDNA sequences (e.g. no introns in the transgenes).
1002521 Additionally, the expression cassette can comprise at least 4000 nucleotides, at least 5000 nucleotides, at least 10,000 nucleotides, at least 20,000 nucleotides, at least 30,000 nucleotides, at least 40,000 nucleotides, or at least 50,000 nucleotides. In some embodiments, the expression cassette can comprise any range of from about 4000 to about 10,000 nucleotides from about 10,000 to about 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene in the range of from about 500 to about 50,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene in the range of from about 500 to about 75,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene that is in the range of from about 500 to about 10,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene that is in the range of from about 1000 to about 10,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene that is in the range of from about 500 to about 5,000 nucleotides in length. In some embodiment, the DNA molecules provided herein do not have the size limitations of encapsidated AAV vectors, thus enabling delivery of a large-size expression cassette to provide efficient transgene. In certain embodiments, the DNA
molecules provided herein comprise expression cassette equal to or larger than the size of any natural AAV genome.
1002531 The expression cassette can have various positions relative to the inverted repeat.
In some embodiments, the expression cassette is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides apart from the inverted repeat. In certain embodiments, the expression cassette is at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6, at least kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 1.5kb, or at least 2 kb apart from the inverted repeat. In other embodiments, the expression cassette is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides apart from the inverted repeat. In further embodiments, the expression cassette is about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6, about kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.5kb, or about 2 kb apart from the inverted repeat. In one embodiment, the inverted repeat in this paragraph is the first inverted repeat as described in Sections 3 and 5.4 (including 5.4.1). In another embodiment, the inverted repeat in this paragraph is the second inverted repeat as described in Sections 3 and 5.4 (including 5.4.1) In yet another embodiment, the inverted repeat in this paragraph is both the first and the second inverted repeat as described in Sections 3 and 5.4 (including 5.4.1) 1002541 The various embodiments described in this Section (Section 5.4.3) with nicking endonucleases and/or restriction sites for nicking endonucleases are additionally provided with nicking endonucleases replaced by programmable nicking enzyme and restriction sites replaced by targeting sites for programmable nicking enzyme. The programmable nicking enzymes and their targeting sites for this paragraph and this Section (Section 5.4.3) have been provided in Section 5.3.4.
5.4.5 Viral DNA Sequence Features Absent in the DNA Molecules Provided Herein 1002551 As further described in Sections 3, 5.2, 5.4.1, 5.4.2, 5.4.3, 5.4.6, 5.4.7 and 5.5, the DNA molecules provided can be produced either synthetically or recombinantly with or without certain sequence elements or features. As such, certain suitable and desired sequence features or elements can be included in the DNA molecules provided herein or excluded from the DNA molecules provided herein. The corresponding methods for making such DNA
molecules including or excluding the sequence features or elements are also provided herein as described by applying the methods of 5.2 with the DNA molecules of 5.4, which can produce various DNA molecules described in 51 1002561 As described in Sections 3, 5.4.1, 5.6, and 6, such DNA sequence elements or features that can be excluded from the DNA molecules provided herein can be a viral replication-associated protein binding sequence ("RABS"), which refers to a DNA sequence to which viral DNA replication-associated proteins and isoforms thereof, encoded by Parvoviridae genes Rep and NS1 can bind. A RABS refers to a nucleotide sequence that includes both the nucleotide sequence recognized by a Rep or NS1 protein (for replication of viral nucleic acid molecules) and the site of specific interaction between the Rep or NS1 protein and the nucleotide sequence. A RABS can be a sequence of 5 nucleotides to 300 nucleotides. In some embodiments of the DNA molecules provided herein including those provided in this Section 5.4.5, the RABS can be a sequence of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, or at least 400 nucleotides. In some other embodiments, the RABS can be a sequence of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, or about 400 nucleotides. In some further embodiments, any embodiment of the DNA molecules lacking an RABS
described in this paragraph can be combined with any methods or DNA molecules provided herein including those provided in Sections 3, 52, 54, 55, and 6 1002571 Alternatively, the DNA molecules provided herein, including those in Sections 3, 5.2, 5.4, 5.5, and 6, can lack a functional RABS by functionally inactivating the RABS
sequence present in the DNA molecules with mutations, insertions, deletions (including partial deletions or truncations), such that the RABS can no longer serve as a recognition and/or binding site for the Rep protein or NS1 protein. As such, in some embodiments of the DNA molecules provided herein, including those in Sections 3, 5.2, 5.4, 5.5, and 6, the DNA
molecule comprise a functionally inactivated RABS. Such functional inactivation can be assess by measuring and comparing the binding between the Rep or NS1 protein and the DNA molecules comprising the functionally inactivated RABS with that between the Rep or NS1 proteins and a reference molecule comprising the wild type (wt) RBS or NSBE
sequences (e.g. the same DNA molecule but with wt RBS or wt NSBE sequences).
Such binding can be determined by any binding measurements known and used in the field of molecular biology, for example, chromatin immunoprecipitation (ChIP) assays, DNA
electrophoretic mobility shift assay (EMSA), DNA pull-down assays, or Microplate capture and detection assays, as further described in Matthew J. Guille & G. Geoff Kneale, Molecular Biotechnology 8:35-52 (1997); Bipasha Dey et al., Mol Cell Biochem. 2012 Jun;365(1-2):279-99, both of which are hereby incorporated in their entireties by reference. In one embodiment, the binding between the RAPs and the functionally inactivated RABS
in the DNA molecule is at most 0.001%, at most 0.01%, at most 0.1%, at most 1%, at most 1.5%, at most 2%, at most 2.5%, at most 3%, at most 3.5, at most 4%, at most 4.5%, at most 5%, at most 5.5%, at most 6%, at most 6.5%, at most 7%, at most 7.5%, at most 8%, at most 8.5%, at most 9%, at most 9.5%, or at most 10%, compared to the binding between the RAPs and the wild type RBS or NSBE in a reference DNA molecule (e.g. the same DNA
molecule but with a wild type RBS or NSBE sequence). In another embodiment, the binding between the RAPs and the functionally inactivated RABS in the DNA molecule is about 0.001%, about 0.01%, about 0.1%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%, compared to the binding between the RAPs and the wild type RBS in a reference DNA molecule (e.g. the same DNA
molecule but with a wt RBS or NSBE sequence). In yet another embodiment, the binding between the RAPs and the functionally inactivated RABS in the DNA molecule is 0.001%, 0.01%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%, compared to the binding between the RAPs and the wild type RABS
in a reference DNA molecule (e.g. the same DNA molecule but with a wt RBS or NSBE
sequence).
1002581 Furthermore, the DNA molecules provided herein, including those in Sections 3, 5.2, 5.4, 5.5, and 6, can lack a functional RAPs or viral capsid encoding sequence by functionally inactivating the Rep protein, NS1 or viral capsid encoding sequence present in the DNA molecules with mutations, insertions, deletions (including partial deletions or truncations), such that the RAPs or viral capsid encoding sequence can no longer functionally express the Rep protein, NS1 protein or viral capsid protein. Such functional inactivating mutations, insertions, or deletions can be achieved, for example, by using mutations, insertions, and/or deletions to shift the open reading frame of Rep protein or viral capsid encoding sequence, by using mutations, insertions, and/or deletions to remove the start codon, by using mutations, insertions, and/or deletions to remove the promoter or transcription initiation site, by using mutations, insertions, and/or deletions to remove the RNA polymerase binding sites, by using mutations, insertions, and/or deletions to remove the ribosome recognition or binding sites, or other means known and used in the field.
1002591 In one embodiment, the DNA molecule comprise an RBS inactivated by mutation.
In one embodiment, the DNA molecule comprise an RBS inactivated by a mutation of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the RBS. In another embodiment, the DNA molecule comprise an RBS inactivated by a mutation of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the nucleotides in the RBS. In a further embodiment, the DNA
molecule comprise an RBS inactivated by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the RBS. In yet another embodiment, the DNA
molecule comprise an RBS inactivated by a deletion of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%
of the nucleotides in the RBS. In some embodiments, the deletion of the preceding sentence is an internal deletion, a deletion from the 5' end, or a deletion from the 3' end. In some embodiments, the deletion of this paragraph can be any combination of internal deletions, deletion from the 5' end, and/or deletions from the 3' end. In certain embodiments, the DNA
molecule comprise an RBS inactivated by a deletion of the entire RBS
sequences. In some additional embodiments, the DNA molecule comprise an RBS inactivated by a partial deletion of the RBS sequences.
1002601 In one embodiment, the DNA molecule comprise an NBSE inactivated by mutation. In one embodiment, the DNA molecule comprise an NSBE inactivated by a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the NSBE.
In another embodiment, the DNA molecule comprise an NSBE inactivated by a mutation of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the nucleotides in the NSBE. In a further embodiment, the DNA molecule comprise an NSBE inactivated by a deletion of 1, 2, 3, 4, 5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the NSBE. In yet another embodiment, the DNA molecule comprise an NSBE inactivated by a deletion of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the nucleotides in the NSBE. In some embodiments, the deletion of the preceding sentence is an internal deletion, a deletion from the 5' end, or a deletion from the 3' end. In some embodiments, the deletion of this paragraph can be any combination of internal deletions, deletion from the 5' end, and/or deletions from the 3' end. In certain embodiments, the DNA molecule comprise an NSBE inactivated by a deletion of the entire NSBE sequences. In some additional embodiments, the DNA molecule comprise an NSBE
inactivated by a partial deletion of the NSBE sequences.
1002611 Similarly, DNA sequence elements or features can be included or excluded from any specific regions of the DNA molecules provided herein (including Sections 5.4 and 5.5) or any specific regions of the DNA molecules used in the methods provided herein (including Section 5.2). In one embodiment, the DNA molecule lacks a Rep protein encoding sequence.
In one embodiment, the DNA molecule lacks a NS1 protein encoding sequence. In another embodiment, the DNA molecule lacks a viral capsid protein encoding sequence.
In some embodiments, the expression cassette lacks a Rep protein encoding sequence. In some embodiments, the expression cassette lacks a NS1 protein encoding sequence. In certain embodiments, the expression cassette lacks a viral capsid protein encoding sequence. In a further embodiment, the DNA molecule lacks an RABS. In yet another embodiment, the first inverted repeat lacks an RABS In one embodiment, the second inverted repeat lacks an RABS. In another embodiment, the DNA sequence between the ITR closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat lacks an RABS. In one embodiment, the DNA molecule comprises a functionally inactivated Rep protein encoding sequence. In one embodiment, the DNA molecule comprises a functionally inactivated NS1 protein encoding sequence. In another embodiment, the DNA
molecule comprises a functionally inactivated viral capsid protein encoding sequence.
In some embodiments, the expression cassette comprises a functionally inactivated Rep protein encoding sequence. In some embodiments, the expression cassette comprises a functionally inactivated NS1 protein encoding sequence. In certain embodiments, the expression cassette comprises a functionally inactivated viral capsid protein encoding sequence.
In a further embodiment, the DNA molecule comprises a functionally inactivated RABS. In yet another embodiment, the first inverted repeat comprises a functionally inactivated RABS. In one embodiment, the second inverted repeat comprises a functionally inactivated RABS. In another embodiment, the DNA sequence between the ITR closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat comprises a functionally inactivated RABS.
1002621 Additionally, DNA sequence elements or features can be functionally inactivated from any combination of any specific regions of the DNA molecules provided herein (including Sections 5.4 and 5.5) or any specific regions of the DNA molecules used in the methods provided herein (including Section 5.2). In one embodiment, the first inverted repeat comprises a functionally inactivated RABS and the second inverted repeat comprises a functionally inactivated RABS. In another embodiment, the first inverted repeat comprises a functionally inactivated RABS and the DNA sequence between the ITR closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat comprises a functionally inactivated RABS. In a further embodiment, the second inverted repeat comprises a functionally inactivated RABS and the DNA sequence between the ITR
closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat comprises a functionally inactivated RABS. In yet another embodiment, the first inverted repeat comprises a functionally inactivated RABS, the second inverted repeat comprises a functionally inactivated RBS and the DNA sequence between the ITR
closing base pair of the first inverted repeat and the ITR closing base pair of the second inverted repeat comprises a functionally inactivated RABS.
1002631 As described in Sections 3, 5.4.1, 5.6, and 6, such DNA sequence elements or features that can be excluded from the DNA molecules provided herein can be a terminal resolution site (TRS). A TRS refers to a nucleotide sequence in the inverted repeat of the DNA molecules that includes the nucleotide sequence recognized by a RAP (for replication of viral nucleic acid molecules), the site of specific interaction between the RAP and the nucleotide sequence, and the site of specific cleavage by the endonuclease activity of the RAP protein. Nucleotide sequences of the conserved sites of specific cleavage by the endonuclease activity of the RAP proteins can be determined by DNA nicking assay known and used in the field of molecular biology, for example, gel electrophoreris, fluorophore-based in vitro nicking assays, radioactive in vitro nicking assay, as further described in Xu P, et al 2019. Antimicrob Agents Chemother 63:e01879-18., US20190203229A, both of which are hereby incorporated in their entireties by reference. In some embodiments a TRS can be a nucleotide sequence in the inverted repeat of the DNA molecules that includes the nucleotide sequence recognized by a Rep protein (for replication of viral nucleic acid molecules), the site of specific interaction between the Rep protein and the nucleotide sequence, and the site of specific cleavage by the endonuclease activity of the Rep protein. In one embodiment a TRS can be a nucleotide sequence in the inverted repeat of the DNA molecules that includes the nucleotide sequence recognized by a NS1 protein (for replication of viral nucleic acid molecules), the site of specific interaction between the NS1 protein and the nucleotide sequence, and the site of specific cleavage by the endonuclease activity of the NS1 protein.
A TRS can be a sequence of 5 nucleotides to 300 nucleotides. In some embodiments of the methods provided herein including those provided in this Section 5.4.5, the TRS can be a sequence of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, or at least 400 nucleotides. In some other embodiments, the TRS can be a sequence of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, or about 400 nucleotides. In some further embodiments, any embodiment of the TRS described in this paragraph can be combined with any methods or DNA
molecules provided herein including those provided in Sections 3, 5.2, 5.4, 5.5, and 6.
1002641 Alternatively, the DNA molecules provided herein, including those in Sections 3, 5.2, 5.4, 5.5, and 6, can lack a functional TRS by functionally inactivating the TRS sequence present in the DNA molecules with mutations, insertions, deletions (including partial deletions or truncations), such that the TRS can no longer serve as a recognition and/or binding site for the RAP (i.e. Rep and NS1). As such, in some embodiments of the DNA
molecules provided herein, including those in Sections 3, 5.2, 5.4, 5.5, and 6, the DNA
molecule comprise a functionally inactivated TRS. Such functional inactivation can be assess by measuring and comparing the binding between the RAP (i.e. Rep and NS1) and the DNA molecules comprising the functionally inactivated TRS with that between the RAP and a reference molecule comprising the wild type (wt) TRS sequences (e.g. the same DNA
molecule but with a wt TRS sequence). Such binding can be determined by any binding measurements known and used in the field of molecular biology, for example, chromatin immunoprecipitation (ChIP) assays, DNA electrophoretic mobility shift assay (EMSA), DNA
pull-down assays, or Microplate capture and detection assays, as further described in Matthew J. Guille & G. Geoff Kneale, Molecular Biotechnology 8:35-52 (1997);
Bipasha Dey et al., Mol Cell Biochem. 2012 Jun;365(1-2):279-99, both of which are hereby incorporated in their entireties by reference. In one embodiment, the binding between the RAP (i.e. Rep and NS1) and the functionally inactivated TRS in the DNA
molecule is at most 0.001%, at most 0.01%, at most 0.1%, at most 1%, at most 1.5%, at most 2%, at most 2.5%, at most 3%, at most 3.5, at most 4%, at most 4.5%, at most 5%, at most 5.5%, at most 6%, at most 6.5%, at most 7%, at most 7.5%, at most 8%, at most 8.5%, at most 9%, at most 9.5%, or at most 10%, compared to the binding between the RAP (i.e. Rep and NS1) and the wild type TRS in a reference DNA molecule (e.g. the same DNA molecule but with a wt TRS
sequence) In another embodiment, the binding between the RAP (i e Rep and NS1) and the functionally inactivated TRS in the DNA molecule is about 0.001%, about 0.01%, about 0.1%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%, compared to the binding between the RAP (i.e.
Rep and NS1) and the wild type TRS in a reference DNA molecule (e.g. the same DNA
molecule but with a wt TRS sequence). In yet another embodiment, the binding between the RAP (i.e. Rep and NS1) and the functionally inactivated TRS in the DNA
molecule is 0.001%, 0.01%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%, compared to the binding between the RAP
(i.e. Rep and NS1) and the wild type TRS in a reference DNA molecule (e.g. the same DNA
molecule but with a wt TRS sequence).
1002651 In one embodiment, the DNA molecule comprise a TRS inactivated by mutation.
In one embodiment, the DNA molecule comprise a TRS inactivated by a mutation of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the TRS. In another embodiment, the DNA molecule comprise a TRS inactivated by a mutation of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the nucleotides in the TRS. In a further embodiment, the DNA
molecule comprise a TRS inactivated by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 10, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in the TRS. In yet another embodiment, the DNA
molecule comprise a TRS inactivated by a deletion of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 10%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%
of the nucleotides in the TRS. In some embodiments, the deletion of the preceding sentence is an internal deletion, a deletion from the 5' end, or a deletion from the 3' end. In some embodiments, the deletion of this paragraph can be any combination of internal deletions, deletion from the 5' end, and/or deletions from the 3' end. In certain embodiments, the DNA
molecule comprise a TRS inactivated by a deletion of the entire TRS sequences.
In some additional embodiments, the DNA molecule comprise a TRS inactivated by a partial deletion of the TRS sequences.
1002661 Similarly, DNA sequence elements or features can be included or excluded from any specific regions of the DNA molecules provided herein (including Sections 14 and 55) or any specific regions of the DNA molecules used in the methods provided herein (including Section 5.2). In one embodiment, the DNA molecule lacks a TRS. In yet another embodiment, the first inverted repeat lacks a TRS. In another embodiment, the second inverted repeat lacks a TRS. In a further embodiment, the first inverted repeat lacks a TRS
and the second inverted repeat lacks a TRS.
1002671 Alternatively, TRS sequence elements or features can be functionally inactivated from any specific regions of the DNA molecules provided herein (including Sections 5.4 and 5.5) or any specific regions of the DNA molecules used in the methods provided herein (including Section 5.2). In one embodiment, the DNA molecule comprises a functionally inactivated TRS. In yet another embodiment, the first inverted repeat comprises a functionally inactivated TRS. In another embodiment, the second inverted repeat comprises a functionally inactivated TRS. In a further embodiment, the first inverted repeat comprises a functionally inactivated TRS and the second inverted repeat comprises a functionally inactivated TRS.
1002681 In some specific embodiments, the RBS excluded or functionally inactivated in the DNA molecules provided herein can be any, or any combination of any number, or all of the RBS sequences listed in Table 20.
Table 20: Exemplary RAPs RAPs Corresponding RABS sequences Rep (AAV1,2,7) GCGCGCTCGCTCGCTC

RAPs Corresponding RABS sequences Rep (AAV3) TGCGCACTCGCTCGCTC
Rep (AAV4) GCGCGCTCGCTCACTC
Rep (AAV5) GTTCGCTCGCTCGCTGGCTC
NS1-NSBE1 (B19V) GCCGCCGG
NS 1 -NSBE2 (B 1 9V) GGCGGGAC
NS1-NSBE3 (B19V) TTCCGGTACA
[00269] In one specific embodiment, the DNA molecules lack encoding sequences for any one, or any combination of any number, or all of the RAPs described in the Table of the preceding paragraph. In another specific embodiment, the DNA molecules comprises functionally inactivated sequences encoding for any one, or any combination of any number, or all of the RAPs described in the Table of the preceding paragraph.
[00270] In other specific embodiments, the TRS excluded or functionally inactivated in the DNA molecules provided herein can be any, or any combination of any number, or all of the TRS sequences listed in Table 21.
Table 21: Exemplary RAPs RAP (Virus) Corresponding TRS sequences Rep(AAV1, AAV2, AAV3, AAV4) AGTTGG
Rep(AAV5) AGTGTGGC
NS1 (B19) GACACC
NS1 (HBOV) CTATATCT
NS 1 (MV1VI) CTWW/TCA (W=AIT) [00271] As the methods provided herein do not need a viral replication step and the DNA
molecules provide herein do not need to be produced or replicated in a virus life cycle, the disclosure provides and a person reading the disclosure would understand that the DNA
molecules provide herein can lack various DNA sequences or features, including those sequences or features provided in this Section (Section 5.4.5). DNA molecules lacking RABS and/or TRS and DNA molecules comprising functionally inactivated RABS
and/or functionally inactivated TRS as provided in this Section 5.4.5 provide at least a major advantage in that the DNA molecules would have no or significantly lower risk of mobilization or replication once administered to a patient when compared with DNA
molecules including such RABS and/or TRS sequences. Risk of mobilization or mobilization risk refers to the risk of the replication defective DNA molecules reverting to replication or production of viral particles in the host that has been administered the DNA
molecules. Such mobilization risk can result from the presence of viral proteins (e.g. Rep proteins, NS1 proteins or viral capsid proteins) expressed by viruses that have infected the same host that has been administered the DNA molecules. Mobilization risk poses a significant safety concern for using the replication defective viral genome as gene therapy vectors, as described for example in Liujiang Song, Hum Gene Ther, 2020 Oct;31(19-20):1054-1067 (incorporated herein in its entirety by reference). Such DNA molecules lacking RBS and/or TRS would have no binding site for viral Rep protein to initiate the replication even if other helper viruses are present in the same host to provide Rep proteins.
1002721 Accordingly, in some embodiments of the DNA molecules provided herein including those in this Section 5.4.5, the DNA molecules without RABS and/or without TRS
have less mobilization risk after administered to a subject or a patient when compared with DNA molecules with RABS and/or with TRS In certain embodiments of the DNA
molecules provided herein including those in this Section 545, the DNA
molecules comprising functionally inactivated RABS and/or functionally inactivated TRS
have less mobilization risk after administered to a subject or a patient when compared with DNA
molecules with RABS and/or with TRS. Such reduction of mobilization risk can be determined as (Pm-Po)/Pm, wherein Pm is the number of viral particles produced from the control DNA molecules with RBS when RAPs are present (e.g. due to the infection of any virus comprising RAPs or engineered expression of RAPs in the same host); Po is the number of viral particles produced from DNA molecules lacking RABS or comprising functionally inactivated as provided herein under comparable conditions in the same host used for the control DNA molecules. Alternatively, such reduction of mobilization risk can be determined as (Pm-Po)/Pm, wherein Pm is the number of viral particles produced from the control DNA molecules with TRS when RAPs are present (e.g. due to the infection of any virus comprising Rep proteins or engineered expression of Rep proteins in the same host); Po is the number of viral particles produced from DNA molecules lacking TRS or comprising functionally inactivated TRS as provided herein under comparable conditions in the same host used for the control DNA molecules. Additionally, such reduction of mobilization risk can be determined as (Pm-Po)/Pm, wherein Pm is the number of viral particles produced from the control DNA molecules with RABS and with TRS when RAPs are present (e.g. due to the infection of any virus comprising Rep proteins, NS1 proteins or engineered expression of Rep proteins in the same host); Po is the number of viral particles produced from DNA
molecules (i) lacking RABS or comprising functionally inactivated RABS and (ii) lacking TRS or comprising functionally inactivated TRS as provided herein under comparable conditions in the same host used for the control DNA molecules. As described in Liujiang Song, Hum Gene Ther, 2020 Oct;31(19-20):1054-1067 (incorporated herein in its entirety by reference), the host used for determining the particle numbers produced can be cells, animals (e.g. mouse, hamster, rate, dog, rabbit, guinea pig, and other suitable mammals), or human.
The disclosure further provides and a person of ordinary skill in the art reading the disclosure would understand that Pm and Po, each as described in this paragraph, can be used also to determine the absolute or relative levels of mobilization. Briefly, in such an assay, the DNA
molecules are transfected into the host cells (e.g. HEK293 cells) or transduced into the host cells by infecting with a viral particle comprising DNA molecules. The host cells are further transfected with Rep protein, NS1 protein or co-infected with another virus expressing the Rep protein or NS1 protein (for example wild type viruses). The host cells are then cultured to produce and release viral particles. Virions are then harvested by collecting both the host cell and the culture media after culturing 48 to 72 hours (e.g. 65 hours) The titer for the viral particles (proxy for Pm and Po) can be determined by a probe-based quantitative PCR
(qPCR) analysis following Benzonase treatment to eliminate nonencapsidated DNA, as described in Song et al., Cytotherapy 2013;15:986-998, which is incorporated in its entirety by reference. An exemplary implementation of such assay is provided in Liujiang Song, Hum Gene Ther, 2020 Oct;31(19-20):1054-1067, which is incorporated herein in its entirety by reference.
1002731 Based on the determination of the reduction of mobilization risk and the mobilization risk levels, in some embodiments of the DNA molecules provided herein including in this Section 5.4.5, the mobilization risk of the DNA molecules when administered to a host is lower than control DNA molecules with RABS and/or with TRS by 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%. In certain embodiments, the mobilization risk of the DNA molecules when administered to a host is lower than control DNA molecules with RABS and/or with TRS by at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%, at least 65%, at least 64%, at least 63%, at least 62%, at least 61%, at least 60%, at least 59%, at least 58%, at least 57%, at least 56%, at least 55%, at least 54%, at least 53%, at least 52%, at least 51%, at least 50%, at least 49%, at least 48%, at least 47%, at least 46%, at least 45%, at least 44%, at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, or at least 20. In other embodiments, the mobilization risk of the DNA molecules when administered to a host is lower than control DNA molecules with RABS and/or with TRS by about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, or about 20%.
1002741 Alternatively, in one embodiment, the DNA molecules provided herein including in this Section 5.4.5, result in no detectable mobilization (e.g. based on the measurement of Po provided in this Section 5.4.5). In another embodiment, the DNA molecules provided herein including in this Section 5.4.5 result in mobilization of no more than 0.0001%, no more than 0.001%, no more than 0.01%, no more than 0.1%, no more than 1%, no more than 1.5%, no more than 2%, no more than 2.5%, no more than 3%, no more than 3.5, no more than 4%, no more than 4.5%, no more than 5%, no more than 5.5%, no more than 6%, no more than 6.5%, no more than 7%, no more than 7.5%, no more than 8%, no more than 8.5%, no more than 9%, no more than 9.5%, or no more than 10%, of the mobilization resulted from a reference DNA molecule (e.g. the same DNA molecule but with a wild type RABS
and/or with wild type TRS sequence). In a further embodiment, the DNA
molecules provided herein including in this Section 5.4.5 result in mobilization of about 0.0001%, about 0.001%, about 0.01%, about 0.1%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%, of the mobilization resulted from a reference DNA molecule (e.g. the same DNA molecule but with a wild type RABS and/or with wild type TRS sequence). In a yet another embodiment, the DNA

molecules provided herein including in this Section 5.4.5 result in mobilization of 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%, of the mobilization resulted from a reference DNA
molecule (e.g. the same DNA molecule but with a wild type RABS and/or with wild type TRS sequence). Such percentage of mobilization can be determined by using the Pm and Po determined as further described in the preceding paragraphs (including the preceding 2 paragraphs).
1002751 As is clear from the descriptions in this Section 5,4.5, the DNA
sequences or features excluded in the DNA molecules provided herein can be combined in any way with any of the methods provided herein (including in Sections 3, 5.2, and 6), any of the DNA
molecules provided herein (including Sections 3, 5.4, and 6), and any of the hairpin-ended DNA molecules provided herein (including Sections 3, 5.5, and 6), and contribute to the functional properties of the DNA molecules as provided herein (including Sections 3, 5.6, and 6).
5.4.6 Vectors such as Plasmids 1002761 The disclosure provides that the DNA molecules can be of various forms. In one embodiment, the DNA molecule provided for the methods and composition herein is a vector. A vector is a nucleic acid molecule that can be replicated and/or expressed in a host cell. Any vectors known to those skilled in the art are provided herein. In some embodiments, the vector can be plasmids, viral vectors, cosmids, and artificial chromosomes (e.g., bacterial artificial chromosomes or yeast artificial chromosomes). In one specific embodiment, the vector is a plasmid. As is clear from the description, when the DNA
molecules are in the form of a vector (including a plasmid), the vector would comprise all the features described herein for the DNA molecules, including those described in Section 3 and this Section (Section 5.4).
1002771 In some embodiments, the vector provided in this Section (Section 5.4.6) can be used for the production of DNA molecules provided in Sections 3 and 5.5, for example by performing the method steps provide din Section 5.2. As such, the vector provided in this Section (Section 5.4.6) (1) comprises the features of the DNA molecules provided in Sections 3 and 5.5, including IRs or ITRs that can form hairpins as described in Sections 5.4.1 and 5.5, expression cassette as described in 5.4.3, and restriction sites for nicking endonucleases or restriction enzymes as described in Sections 5.4.2, 5.3.4, and 5.4.7, and/or (2) lacks the RABS and/or TRS sequences as described in Section 5.4.5. Therefore, the disclosure provides that the vector provided in this Section (Section 5.4.6) can (1) comprise any combination of embodiments of IRs or ITRs that can form hairpins as described in Sections 5.4.1 and 5.5, expression cassette as described in 5.4.3, restriction sites for nicking endonucleases or restriction enzymes as described in Sections 5.4.2 5.3.4, and 5.4.7, and additional features for the vectors provided in this Section (Section 5.4.6), and/or (2) lacks the RABS and/or TRS sequences as described in Section 5.4.5. In some embodiments, a vector can be constructed using known techniques to provide at least the following as operatively linked components in the direction of transcription: (1) a 5' ITR
sequence; (2) an expression cassette comprising a cis-regulatory element, for example, a promoter, inducible promoter, regulatory switch, enhancers and the like; and (3) a 3' IR sequence.
In some embodiments, the expression cassette is flanked by the ITRs comprises a cloning site for introducing an exogenous sequence.
1002781 Specifically, in one embodiment, the DNA molecule is a plasmid.
Plasmid is widely known and used in the art as a vector to replicate or express the DNA
molecules in the plasmid. Plasmid often refers to a double-stranded and/or circular DNA
molecule that is capable of autonomous replication in a suitable host cell. Plasmids provided for the methods and compositions described herein include commercially available plasmids for use in well-known host cells (including both prokaryotic and eukaryotic host cells), as available from various vendors and/or described in Molecular Cloning: A Laboratory Manual, 4th Edition, by Michael Green and Joseph Sambrook, ISBN 978-1-936113-42-2 (2012), which is incorporated herein in its entirety by reference.
1002791 The plasmids described in this Section (Section 5.4.6) can further comprise other features. In some embodiments, the plasmid further comprises a restriction enzyme site (e.g.
restriction enzyme site as described in Sections 5.3.4 and 5.4.2) in the region 5' to the first inverted repeat and 3' to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and the region between the first and second inverted repeats. In certain embodiments, the cleavage with the restriction enzyme at the restriction site described in this paragraph results in single strand overhangs that do not anneal at detectable levels under conditions that favor annealing of the first and/or second inverted repeat (e.g. conditions as described in Section 5.3.5). In some other embodiments, the plasmid further comprises an open reading frame encoding the restriction enzyme recognizing and cleaving the restriction site describe in this paragraph. In certain embodiments, the restriction enzyme site and the corresponding restriction enzyme can be any one of the restriction enzyme site and its corresponding restriction enzyme described in Sections 5.3.4 and 5.4.2. In further embodiment, the expression of the restriction enzyme described in this paragraph is under the control of a promoter. In some embodiments, the promoter described in this paragraph can be any promoter described above in Section 5.4.3.
In other embodiment, the promoter described is an inducible promoter. In certain embodiment, the inducible promoter is a chemically inducible promoter. In further embodiments, the inducible promoter is any one selected from the group consisting of:
tetracycline ON (Tet-On) promoter, negative inducible pLac promoter, alcA , anlyB, hli-3, bphA, catR, cbhl , cre 1 , exylA, gas, glaA, gla 1 , mirl, niiA, qa-2, Stnxyl, tcu- , thiA, vvd, xyl , xyl I ,xylP, xyn I , and ZeaR, as described in Janina Kluge et al, Applied Microbiology and Biotechnology 102: 6357-6372 (2018), which is incorporated herein in its entirety by reference.
1002801 Similarly, in certain embodiments, the plasmid can further comprise a fifth and a sixth restriction site for nicking endonuclease (e.g. restriction site for nicking endonuclease as described in Sections 5.3.4 and 5.4.2) in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are: a.) on opposite strands; and b.) create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intra-molecularly under conditions that favor annealing of the first and/or second inverted repeat (e.g. conditions as described in Section 5.3.5). As is clear from the description of Section 5.3.4, incubation with nicking endonucleases will result in a fifth nick corresponding to the fifth restriction site for the nicking endonuclease and a sixth nick corresponding to the sixth restriction site for the nicking endonuclease. The disclosure provides that the fifth and sixth nick can have various relative positions between them. In one embodiment, the fifth and the sixth nick are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart. In some embodiments, as the ssDNA overhang between fifth and sixth nick does not anneal at detectable levels inter- or intra-molecularly under conditions that favor annealing of the first and/or second inverted repeat, the ssDNA overhang resulted from fifth and sixth nick has a lower melting temperature than the ssDNA overhangs described in Sections 5.3.3 and 5.4.2. In certain embodiments, the ssDNA overhang resulted from fifth and sixth nick is shorter than the ssDNA overhangs described in Sections 5.3.3 and 5.4.2. In other embodiments, the ssDNA overhang resulted from fifth and sixth nick has a lower percentage of G-C content than the ssDNA overhangs described in Sections 5.3.3 and 5.4.2.
In some specific embodiments, the ssDNA overhang resulted from fifth and sixth nick is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
1002811 In certain embodiments, the plasmid can further comprise 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19 or more restriction sites for nicking endonuclease (e.g. restriction site for nicking endonuclease as described in Sections 5.3.4 and 5.4.2) in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the additional restriction sites for nicking endonuclease are: a.) on opposite strands; and b.) create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intra-molecularly under conditions that favor annealing of the first and/or second inverted repeat (e.g. conditions as described in Section 5.3.5).
The disclosure provides that the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat, can have various relative positions between them. In one embodiment, the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat, are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart. In some embodiments, as the ssDNA overhang between the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat does not anneal at detectable levels inter- or intra-molecularly under conditions that favor annealing of the first and/or second inverted repeat, the ssDNA overhang resulted from the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat has a lower melting temperature than the ssDNA
overhangs described in Sections 5.3.3 and 5.4.2. In certain embodiments, the ssDNA
overhang resulted from the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat is shorter than the ssDNA overhangs described in Sections 5.3.3 and 5.4.2. In other embodiments, the ssDNA overhang resulted from the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat has a lower percentage of G-C content than the ssDNA overhangs described in Sections 5.3.3 and 5.4.2. In some specific embodiments, the ssDNA overhang resulted from the nicks in the region 5' to the first inverted repeat and 3' to the second inverted repeat is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
1002821 As described above in Sections 5.3.4 and 5.4.2, in various embodiments, the first, second, third, and fourth restriction sites for nicking endonuclease can be the target sequences for the same or different nicking endonucleases. Similar, in certain embodiments, the fifth and sixth restriction sites for nicking endonuclease can be target sequences for the same or different nicking endonucleases. In some embodiments, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease provided for the DNA
molecules as described in Sections 3 and 5.3.4 and this Section 5.4 can be all for target sequences for the same nicking endonuclease. Alternatively, in other embodiments, the first, second, third, fourth, fifth, and sixth numbering? restriction sites for nicking endonucleases are target sequences for two different nicking endonucleases, including all possible combinations of arranging the six sites for two different nicking endonuclease target sequences (e.g. the first restriction site for the first nicking endonuclease and the rest for the second nicking endonuclease, the first and second restriction sites for the first nicking endonuclease and the rest for the second nicking endonuclease, etc.). Additionally, in certain embodiments, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonucleases are target sequences for three different nicking endonucleases, including all possible combinations of arranging the six sites for three different nicking endonucl ease target sequences Furthermore, in some embodiments, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are target sequences for four different nicking endonucleases, including all possible combinations of arranging the six sites for four different nicking endonuclease target sequences. Additionally, in some embodiments, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are target sequences for five different nicking endonucleases, including all possible combinations of arranging the six sites for five different nicking endonuclease target sequences. Furthermore, in some embodiments, the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are target sequences for six different nicking endonucleases.
[00283] In some embodiments, the one or more of the nicking endonuclease sites described in the preceding paragraph are a target sequence of an endogenous nicking endonuclease. In some specific embodiments, the plasmid further comprises an ORF
encoding a nicking endonucl ease that recognizes one or more of the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph. In one specific embodiment, the plasmid further comprises two ORFs encoding two nicking endonucl eases that recognize two or more of the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph.
In another specific embodiment, the plasmid further comprises three ORFs encoding three nicking endonucleases that recognize three or more of the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph. In yet another specific embodiment, the plasmid further comprises four ORFs encoding four nicking endonucleases that recognize four or more of the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph. In a further specific embodiment, the plasmid further comprises five ORFs encoding five nicking endonucleases that recognize five or more of the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph. In one specific embodiment, the plasmid further comprises six ORFs encoding six nicking endonucleases that each recognizes the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease described in this Section (Section 5.4.6) including the preceding paragraph. In certain embodiments, the expression of the one or more nicking endonucleases described in this paragraph is under the control of a promoter.
In some embodiments, the expression of the one or more nicking endonucleases described in this paragraph is under the control of an inducible promoter. In some specific embodiments, the inducible promoter can be any inducible promoter described above in this Section (Section 5.4.6).
1002841 In some embodiments, the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease can be any one described in Sections 3, 5.3.4 and 5.4.2. In certain specific embodiment, the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALw1; N. BstNBI; N. BspD6I; Nb. Mva1269I; Nb. BsrDI;
Nt. BtsI;
Nt. BsaI, Nt. Bpul0I; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI. In some embodiments, the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease can be any one described in Sections 3, 5.3.4 and 5.4.2.
In certain specific embodiment, the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N.
BspD6I;
Mva1269I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpul0I; Nt. BsmBI; Nb. BbvCI; Nt.
BbvCI; or Nt. BspQI.
1002851 In some embodiments, the DNA molecules for the methods and composition provided herein (e.g. as provided in Section 3 and this Section (Section 5.4)) can be linear, non-circular DNA molecules.
1002861 In some embodiments, a vector for the methods and composition provided herein comprises any one or more features described in this Section (Section 5.4.6), in various permutations and combinations. In certain embodiments, a plasmid for the methods and composition provided herein comprises any one or more features described in this Section (Section 5.4.6), in various permutations and combinations.
1002871 The various embodiments described in this Section (Section 5.4.6) with nicking endonucleases and/or restriction sites for nicking endonucleases are additionally provided with nicking endonucleases replaced by programmable nicking enzyme and restriction sites replaced by targeting sites for programmable nicking enzyme. The programmable nicking enzymes and their targeting sites for this paragraph and this Section (Section 5.4.3) have been provided in Section 5.3.4.
5.4.7 DNA Molecules With Less Than 4 Restriction Sites for Nicking Endonucleases and DNA Molecules With Less Than 4 Target Sites for Programmable Nicking Enzymes 1002881 In one additional aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second restriction site for nicking endonuclease and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease and the second restriction site for restriction enzyme is more distal to expression cassette than the second restriction site for nicking endonuclease.
1002891 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second restriction site for nicking endonuclease and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease and the second restriction site for restriction enzyme is more distal to expression cassette than the second restriction site for nicking endonuclease.
1002901 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second restriction site for nicking endonuclease and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease and the second restriction site for restriction enzyme is more distal to expression cassette than the second restriction site for nicking endonuclease.
1002911 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second restriction site for nicking endonuclease and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease and the second restriction site for restriction enzyme is more distal to expression cassette than the second restriction site for nicking endonuclease.

Additionally, in one aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3);
and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third restriction site for nicking endonuclease 1002931 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2);
ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third restriction site for nicking endonuclease.
1002941 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.33, 5.3.4 and 5 4 2); ii) an expression cassette (e.g. as described in Section 5 4 3);
and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third restriction site for nicking endonuclease.
1002951 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 54.2);
ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third restriction site for nicking endonuclease.
1002961 Additionally, in one aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second and a third restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease.
1002971 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette encoding for GDE (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease.
1002981 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette encoding for GDE (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease 1002991 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first restriction site for nicking endonuclease and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette encoding for GDE (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first restriction site for nicking endonuclease.
1003001 In one additional aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by the programmable nicking enzyme and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette encoding for GDE (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second target site for the guide nucleic acid for programmable nicking enzyme and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by the programmable nicking enzyme and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme and the second restriction site for restriction enzyme is more distal to expression cassette than the second target site for the guide nucleic acid for programmable nicking enzyme 1003011 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette encoding for (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a second target site for the guide nucleic acid for programmable nicking enzyme and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme and the second restriction site for restriction enzyme is more distal to expression cassette than the second target site for the guide nucleic acid for programmable nicking enzyme.
1003021 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second target site for the guide nucleic acid for programmable nicking enzyme and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 533, 5 3 4 and 5 4 2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme and the second restriction site for restriction enzyme is more distal to expression cassette than the second target site for the guide nucleic acid for programmable nicking enzyme.
1003031 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second target site for the guide nucleic acid for programmable nicking enzyme and a second restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme and the second restriction site for restriction enzyme is more distal to expression cassette than the second target site for the guide nucleic acid for programmable nicking enzyme.
1003041 Additionally, in one aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a third target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third target site for the guide nucleic acid for programmable nicking enzyme.
1003051 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g as described in Sections 5.3.3, 5.3.4 and 5.4.2);
ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third target site for the guide nucleic acid for programmable nicking enzyme.
1003061 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g.
as described in Section 5.4.1), wherein a third target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third target site for the guide nucleic acid for programmable nicking enzyme.
1003071 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first and a second target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the first inverted repeat such that nicking by programmable nicking enzyme results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g as described in Sections 5.3.3, 5.3.4 and 5.4.2);
ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a third target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the second inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the third target site for the guide nucleic acid for programmable nicking enzyme.
1003081 Additionally, in one aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme.
1003091 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme.
1003101 In yet another aspect, provided herein is a double-stranded DNA
molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme.
1003111 In a further aspect, provide herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: i) a first inverted repeat (e.g. as described in Section 5.4.1), wherein a first target site for the guide nucleic acid for programmable nicking enzyme and a first restriction site for restriction enzyme are arranged in the opposite ends and in proximity of the first inverted repeat such that nicking by programmable nicking enzyme and restriction enzyme cleavage result in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2); ii) an expression cassette (e.g. as described in Section 5.4.3); and iii) a second inverted repeat (e.g. as described in Section 5.4.1), wherein a second and a third target site for the guide nucleic acids for programmable nicking enzyme are arranged on opposite strands in proximity of the second inverted repeat such that nicking by programmable nicking enzyme results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat (e.g. as described in Sections 5.3.3, 5.3.4 and 5.4.2), wherein the first restriction site for restriction enzyme is more distal to expression cassette than the first target site for the guide nucleic acid for programmable nicking enzyme.
1003121 The DNA molecules provided in this Section (Section 5.4.7) comprise various features or have various embodiments as described in this Section (Section 5.4.7), which features and embodiments are further described in the various subsections below: the embodiments for the inverted repeats, including the first inverted repeat and/or the second inverted repeat, are described in Section 5.4.1, the embodiments for the restriction enzymes, nicking endonucleases, and their respective restriction sites are described in Section 5.4.2, the embodiments for the programmable nicking enzymes and their target sites are described in Section 5.3.4, the embodiments for the expression cassette are described in Section 5.4.3, and the embodiments for plasmids and vectors are described in Section 5.4.6. As such, the disclosure provides DNA molecules comprising any permutations and combinations of the various embodiments of DNA molecules and embodiments of features of the DNA
molecules described herein.
1003131 The various embodiments described in this Section (Section 5.4.7) with nicking endonucleases are interchangeable with programmable nicking enzyme and restriction sites for nicking endonucleases are interchangeable with the target sites for programmable nicking enzyme. As such, additional embodiments of any combination resulted by replacing one or more elements of nicking endonucleases with programmable nicking enzyme and/or replacing one or more elements of restriction sites for nicking endonucleases with the target sites for programmable nicking enzyme are provided herein in this Section (Section 5.4.7).
The programmable nicking enzymes and their targeting sites for this paragraph and this Section (Section 5.4.3) have been provided in Section 5.3.4.
5.4.8 Isolated DNA Molecules 1003141 One of the advantages of the methods and DNA molecules provided herein is the purity of the isolated DNA molecules produced in the methods and provided herein, because the DNA molecules provided herein are resistant to exonuclease or other DNA
digestion enzymes and thus can be treated, as described in Section 5.3.6, with such exonuclease or DNA digestion enzymes to remove the DNA contaminants that are susceptible to such treatment. As already described in the paragraphs between the heading of Section 5.4 and the heading of Section 5.4.1, the DNA molecules provided herein including in Sections 3, 5.2, 5.4, 5.5, and 6 can be isolated DNA molecules of various purity. Furthermore, the disclosure provides and a person of ordinary skill in the art would understand that the DNA molecules provided herein including in Sections 3, 5.2, 5.4, 5.5, and 6 can be free of certain general DNA contaminants, free of certain specific DNA contaminants, or both free of certain general DNA contaminants and free of certain specific DNA contaminants.
1003151 Accordingly, in one embodiment, the isolated DNA molecules are free of fragments of the DNA molecules. In another embodiment, the isolated DNA
molecules are free of nucleic acid contaminants that are not fragments of the DNA molecules.
In a further embodiment, the isolated DNA molecules are free of baculoviral DNA. In one embodiment, the isolated DNA molecules are free of fragments of the DNA molecules and free of nucleic acid contaminants that are not fragments of the DNA molecules. In another embodiment, the isolated DNA molecules are free of fragments of the DNA molecules and free of baculoviral DNA. In a further embodiment, the isolated DNA molecules are free of baculoviral DNA
and free of nucleic acid contaminants that are not fragments of the DNA
molecules. In yet another embodiment, the isolated DNA molecules are free of fragments of the DNA
molecules, free of baculoviral DNA, and free of nucleic acid contaminants that are not fragments of the DNA molecules.
1003161 Specifically, in one embodiment, the fragments of the DNA molecules are no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%, no more than 19%, no more than 20%, no more than 21%, no more than 22%, no more than 23%, no more than 24%, no more than 25%, no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%, no more than 41%, no more than 42%, no more than 43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, or no more than 50% of the isolated DNA molecules.
In another embodiment, the fragments of the DNA molecules are less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%, less than 21%, less than 22%, less than 23%, less than 24%, less than 25%, less than 26%, less than 27%, less than 28%, less than 29%, less than 30%, less than 31%, less than 32%, less than 33%, less than 34%, less than 35%, less than 36%, less than 37%, less than 38%, less than 39%, less than 40%, less than 41%, less than 42%, less than 43%, less than 44%, less than 45%, less than 46%, less than 47%, less than 48%, less than 49%, or less than 50% of the isolated DNA molecules. In yet another embodiment, the fragments of the DNA
molecules are about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the isolated DNA
molecules.
1003171 Additionally, in one embodiment, the nucleic acid contaminants that are not fragments of the DNA molecules are no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%, no more than 19%, no more than 20%, no more than 21%, no more than 22%, no more than 23%, no more than 24%, no more than 25%, no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%, no more than 41%, no more than 42%, no more than 43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, or no more than 50% of the isolated DNA molecules. In another embodiment, the nucleic acid contaminants that are not fragments of the DNA molecules are less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%, less than 21%, less than 22%, less than 23%, less than 24%, less than 25%, less than 26%, less than 27%, less than 28%, less than 29%, less than 30%, less than 31%, less than 32%, less than 33%, less than 34%, less than 35%, less than 36%, less than 37%, less than 38%, less than 39%, less than 40%, less than 41%, less than 42%, less than 43%, less than 44%, less than 45%, less than 46%, less than 47%, less than 48%, less than 49%, or less than 50% of the isolated DNA
molecules. In yet another embodiment, the nucleic acid contaminants that are not fragments of the DNA
molecules are about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the isolated DNA
molecules.
1003181 In addition, in one embodiment, the baculoviral DNA are no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%, no more than 19%, no more than 20%, no more than 21%, no more than 22%, no more than 23%, no more than 24%, no more than 25%, no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%, no more than 41%, no more than 42%, no more than 43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, or no more than 50% of the isolated DNA molecules. In another embodiment, the baculoviral DNA are less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%, less than 21%, less than 22%, less than 23%, less than 24%, less than 25%, less than 26%, less than 27%, less than 28%, less than 29%, less than 30%, less than 31%, less than 32%, less than 33%, less than 34%, less than 35%, less than 36%, less than 37%, less than 38%, less than 39%, less than 40%, less than 41%, less than 42%, less than 43%, less than 44%, less than 45%, less than 46%, less than 47%, less than 48%, less than 49%, or less than 50% of the isolated DNA molecules. In yet another embodiment, the baculoviral DNA are about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%
of the isolated DNA molecules.
1003191 The various embodiments the isolated DNA molecules provided herein of various purities with respect to the specific contaminants as described in the preceding paragraphs (e.g. fragments of the DNA molecules, nucleic acid contaminants that are not fragments of the DNA molecules, and/or baculoviral DNA) of this Section 5.4.8 are not mutually exclusive and thus can be combined in various combinations by selecting and combining any embodiments provided in the list of the preceding paragraphs of this Section 5.4.8.
Furthermore, the isolated DNA molecules provided in this Section 5.4.8 and those in the paragraphs between the heading of Section 5.4 and the heading of Section 5.4.1 can also be combined in various combinations by selecting and combining any suitable embodiments provided in the list described therein.
5.5 Hairpin-ended DNA Molecules The disclosure provides that the hairpin-ended DNA molecules of this Section (Section 5.5) can be produced by performing the method steps described in Section 5.2 (including Sections 5.3.3, 5.3.4, and 5.3.5) on DNA molecules provided in Section 5.4. As such, the hairpin-ended DNA molecules of this Section (Section 5.5) can (1) comprise the various features of the DNA molecules provided in Sections 3 and 5.4, including IRs or ITRs that can form hairpins as described in Section 5.4.1 and this Section (Section 5.5), specific sequences, origins, and identities of IRs or ITRs as described in Section 5.4.1 and this Section (Section 5.5), expression cassette as described in 5.4.3, restriction sites for nicking endonucleases or restriction enzymes as described in Sections 5.4.2, 5.3.4, and 5.4.7, and the targeting sites for programmable nicking enzymes as described in Section 5.3.4, and/or (2) lacks the RABS and/or TRS sequences as described in Section 5.4.5. Therefore, the disclosure provides that the hairpin-ended DNA molecules of this Section (Section 55) can (1) comprise any combination of embodiments of IRs or ITRs that can form hairpins as described in Sections 5.4.1 and this Section (Section 5.5), expression cassette as described in 5.4.3, restriction sites for nicking endonucleases or restriction enzymes as described in Sections 5.4.2, 5.3.4, and 5.4.7, the targeting sites for programmable nicking enzymes as described in Section 5.3.4, and additional features for the vectors provided in this Section (Section 5.5), and /or (2) lacks the RABS and/or TRS sequences as described in Section 5.4.5.
1003211 As is clear from the descriptions, the ITRs or the hairpinned ITRs in the hairpin-ended DNA molecules provided in this Section (Section 5.5) can be formed from the ITRs or IRs provided above in Sections 3 and 5.4.1, for example upon performing the method steps described in Sections 3, 5.3.3, 5.3.4, and 5.3.5. Accordingly, in some embodiments, the two ITRs or the two hairpinned ITRs in the hairpin-ended DNA molecules provided in this Section (Section 5.5) can comprise any embodiments of the IRs or ITRs provided in Sections 3 and 5.4.1 and additional embodiments provided in this Section (Section 5.5), in any combination.
1003221 In one aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: a.) a first hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)); b.) a nick of the bottom strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); c.) an expression cassette (e.g. as described 5.4.3 and this Section (Section 5.5)); d.) a nick of the bottom strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); and e.) a second hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)).
1003231 In another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: a.) a first hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)); b.) a nick of the top strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); c.) an expression cassette (e.g. as described 5.4.3 and this Section (Section 5.5)); d.) a nick of the top strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); and e.) a second hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)).
1003241 In yet another aspect, provided herein is a double strand DNA molecule comprising in 5' to 3' direction of the top strand: a.) a first hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)); b.) a nick of the bottom strand (e.g.
as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); c.) an expression cassette (e.g as described 5.4.3 and this Section (Section 5.5)); d.) a nick of the top strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); and e.) a second hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)).
1003251 In a further aspect, provided herein is a double strand DNA
molecule comprising in 5' to 3' direction of the top strand: a.) a first hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)); b.) a nick of the top strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); c.) an expression cassette (e.g. as described 5.4.3 and this Section (Section 5.5)); d.) a nick of the bottom strand (e.g. as described in Sections 5.3.4 and 5.4.2, and this Section (Section 5.5)); and e.) a second hairpinned inverted repeat (e.g. as described in Section 5.4.1 and this Section (Section 5.5)).
1003261 The secondary structure is formed based on conformations (e.g.
domains) that include base pair stacking, stems, hairpins, bulges, internal loops and multi-branch loops. A
domain-level description of IRs represents the strand and formed complexes in terms of domains rather than specific nucleotide sequences. At the sequence level, each domain is assigned a particular nucleotide sequence or motif, and its complement's sequence is determined by Watson¨Crick base pairing. This spans the full range of binding between any pair of complementary nucleotides, including G-T wobble base pairs. The overall set of bound (e.g. base paired) and unbound domains form a unimolecular complex and exhibit various secondary structure In some embodiments, hairpins can have a base-paired stem and a small loop of unpaired bases. In certain embodiments, the presence of interweaved non-palindromic polynucleotides sections in the polynucleotide sequence can lead to unpaired nucleotides known as bulges. Bulges can have one or more nucleotides and are classified in different types depending on their location: in the top strand (bulge), in both strands (internal loop) or at a junction. The collection of these base pairs constitutes the secondary structure of DNA, which occur in its three-dimensional structure.
1003271 A domain-level description for the DNA molecules provided herein are also provided to represent multiple strands and their complexes in terms of domains rather than specific nucleotide sequences. In some embodiments, domains (e.g. sequences motifs) of interacting single stranded DNA strands can exhibit particular secondary structures on a single strand level that can interact with other DNA strands and in some cases take on a hybridized structure when a first strand is bound to a complementary domain on a second strand to form a duplex. Interactions of different DNA strands that generate new complexes or changes in secondary structure can be viewed as "reactions." Additional unimolecular and bimolecular reactions are also possible at the sequence level. Poor sequence design can lead to sequence-level structures or interactions (e.g. multiple domains of complimentary in the expression cassette) that interfere with the intended reactions of a system comprising one or more DNA molecules provided herein. Undesired interactions can be avoided by design, resulting in reliable and predictable secondary structure formation.
1003281 The disclosure provides that the underlying forces leading to the secondary structure of DNA are governed by hydrophobic interactions that underlie thermodynamic laws and the overall conformation may be influenced by physicochemical conditions. An exemplary list of factors determining equilibrium state include the type of solvent, chemical agents crowding, salt concentrations, pH and temperature. While free energy change parameters and enthalpy change parameters derived from experimental literature allow for a prediction of conformation stability, the overall three-dimensional structures of the hairpin formed from the IR sequences, as usual in statistical mechanics, corresponds to an ensemble of molecular conformations, not just one conformation. Predominant conformations cam transition as the physical or chemical conditions (e.g. salts, pH or temperature) are permutated.
1003291 "Stem domain" or "stem" refers to a self-complementary nucleotide sequence of the overhang strand that will form Watson-Crick base pairs. The stem comprises primarily Watson-Crick base pairs formed between the two antiparallel stretches of DNA
pairs and can be a right-handed helix In one embodiment, the stem comprises the stretch of self-complimentary DNA sequence in a palindromic sequence.
1003301 "Primary stem domain" or -primary stem" refers to the part of self-complementary or reverse complement nucleotide sequences of the ITR that is most proximal to the expression cassette or the non-ITR sequences of the DNA molecule. In one embodiment, the primary stem domain is the self-complimentary stretch of a palindromic sequence that forms the termini of the DNA molecules provided herein and is covalently linked to the non-ITR sequences flanked by the ITRs. The primary stem encompasses both the start as well as the end of an IR sequence. In certain embodiments, the primary stems range in length from 1 to 100 or more bp. The lengths of primary stem region have an effect on denature/renature kinetics. In some specific embodiments, the primary stem region have at least approximately 4 and 25 nucleotides to ensure thermal stability. In other specific embodiments, the primary stem region have about 4 and 25 nucleotides to ensure thermal stability. On the other hand, the inverted repeat domains may be of any length sufficient to maintain an approximate three dimensional structure at physiological conditions.
1003311 "Loop" or "loop domain" refers to the region of unpaired nucleotides in an IR or ITR that is not a turning point and not in a stem. In some embodiments, a loop domain is found at the apex of the IR structure. The loop domain can serve as the region in which the local directionality of the DNA strand is reversed to afford the two antiparallel strands of the originating stem. Because of steric repulsion, in certain embodiments, a loop comprises a minimum of two nucleotides to make a turn in a DNA hairpin. In other embodiments, a loop comprises four nucleotides or more. In yet other embodiments, a loop comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides. In some further embodiments, a loop comprises about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides. The loop follows a self-complementary sequence of a stem and serves to connect the further nucleotides to the stem domain. In some embodiments, a loop comprise a sequence of oligonucleotides that does not form contiguous duplex structure with other nucleotides in the loop sequence or other elements of the ITR
(e.g., the loop remains in flexible, single-stranded form). In one embodiment, the loop sequence that does not form a duplex with other nucleotides in the loop sequence is a series of identical bases (e.g. AAAAAAAA, CCCCCCCC, GGGGGGG or TTTTTTTT). In one embodiment, the loop contains between 2 and 30 nucleotides. In a further embodiment, the loop domain contains between 2 and 15 nucleotides. In yet a further embodiment, the loop comprises a mixture of nucleotides.
1003321 As used herein, the term "hairpin" refers to any DNA structure as well as the overall DNA structure, including secondary or tertiary structure, formed from an IR or ITR
sequence. As used herein, a "hairpinned" DNA molecule refers to a DNA molecule wherein one or more hairpins has formed in the DNA molecule. In one embodiment, a hairpin comprises a complementary stem and a loop. A hairpin in its simplest form consists of a complementary stem and a loop. A structure encompassing stems and loops are referred to as -stem-loop," -stem loop," or -SL." In another embodiment, a hairpin consists of a complementary stem and a loop. "Branched hairpin" refers to a subset of hairpin that has multiple stem-loops that form branch structures (e.g. as depicted in FIG. 1).
An IR or ITR
after forming hairpin can be referred to as hairpinned ITR or IR. A "hairpin-ended" DNA
molecule refers to a DNA molecule wherein a hairpin has formed at one end of the DNA
molecule or a hairpin has formed at each of the 2 end of the DNA molecule.
1003331 "Turning point" or "apex" refers to the region of unpaired nucleotides at the spatial end of the ITR. The turning point serves as the region in which the global directionality of the DNA strand is reversed to afford the two antiparallel strands of the originating stem. The turning point also marks the point at which the IR or ITR sequence becomes inverted or the reverse compliment.
1003341 In some embodiments, the part of ITR following the primary stem domain, can encode a nucleotide sequence, which in contrast to regular double-stranded DNA, can form non-Watson-Crick-based structural elements when folding on itself, including wobbles and mismatches, and structural defects or imperfections, such as bulges and internal loops (see e.g. FIG. 1). A "bulge" contains one or more unpaired nucleotides on one strand, whereas "internal loops" contain one or more unpaired nucleotides on both top and bottom strands.
Symmetric internal loops tend to distort the helix less than bulges and asymmetric internal loops, which can kink or bend the helix. In some embodiments, the unpaired nucleotides in a stem can engage in diverse structural interactions, such as noncanonical hydrogen bonding and stacking, which lend themselves to additional thermodynamic stability and functional diversity Without being bound by theory, it is thought that the structural diversity of IR
stems and loops leads to complex secondary structures, and functional diversity.
1003351 In some embodiment, a hairpin for the hairpin-ended DNA molecule comprises a primary stem. In one embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5,6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 stems. In another embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 loops. In yet another embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 internal loops. In a further embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bulges. In one embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 branched hairpins. In another embodiment, a hairpin for the hairpin-ended DNA molecule comprises 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 apexes. In a further embodiments, a hairpin for the hairpin-ended DNA molecule comprise any number of stems, branched hairpins, loops, bulges, apexes, and/or internal loops, in any combination.
1003361 In some embodiments, the hairpin structure in the DNA molecules provided herein is formed by a symmetrical overhang. In order to obtain a symmetrical overhang, the modification in the 5' stem region will require a cognate 3' modification at the corresponding position in the stem region so that the modified 5' position(s) can form base pair(s) with the modified 3' position(s). Such modification to form a symmetrical overhang can be performed as described in the present disclosure in combination with the state of the art at the time of filing. For example, by generating a BstNBI restriction site for nicking endonuclease by an insertion of an A at position 23 will require an insertion of T at position 105 with respect to the wt A AV2 ITR (e.g., SEQ ID NO:162).
1003371 In some embodiments, the 5' and 3' hairpinned ITRs from a hairpinned ITR pair can have different reverse complement nucleotide sequences to harbor the antiparallel restriction sites for nicking endonuclease (e.g. 5' ITR such that nicking results in a bottom strand 5' overhang and the 3' ITR such that nicking results in a bottom strand 3' overhang) but still have the same three-dimensional spatial organization such that both ITRs have mutations that result in the same overall 3D shape.
1003381 In some embodiments, hairpinned ITRs for use herein can comprise a modification (e.g., deletion, substitution or addition) of at least 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in any one or more of the regions selected from: the primary stem domain, a stem, a branched hairpin, a loop, a bulge or an internal loop. In one specific embodiment, the nucleotide in a right hairpinned ITR can be substituted from an A to a G, C or T or deleted or one or more nucleotides added; a nucleotide in a left hairpinned ITR can be changed from a T to a G, C or A, or deleted or one or more nucleotides added.
1003391 In some embodiments, the hairpinned ITR of the DNA molecules provided herein can comprise primary stem wherein 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more complementary base pairs are removed from each of the primary stem domains such that the primary stem domain is shorter and has a lower free energy of folding.
Briefly, in such embodiments, if a base is removed in the portion of the primary stem domain, the complementary base pair in primary stem domain is also removed, thereby shortening the overall primary stem domain.
1003401 In some embodiments, the hairpinned ITR of the DNA molecules provided herein can comprise primary stem wherein 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more complementary base pairs are introduced from each of the primary stem domains such that the primary stem domain is longer and has a higher free energy of folding.
Briefly, in such embodiments, if a base is introduced in the portion of the primary stem domain, the complementary base pair in primary stem domain is also introduced, thereby lengthening the overall primary stem domain.
1003411 In some embodiments, the hairpinned ITR of the DNA molecules provided herein can comprise primary stem wherein 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40 or more complementary base pairs are substituted from A or T to G or C from each of the primary stem domains such that the primary stem domain is more G/C rich and has a higher free energy of folding. Briefly, in such embodiments, if a base is substitute (e.g.
T to G) in the portion of the primary stem domain, the complementary base pair in primary stem domain is also substituted (e.g. A to C, thereby increasing the G/C content the overall primary stem domain.
1003421 In some embodiments, a hairpinned ITR sequence in the DNA molecules provide herein can have between 1 and 40 nucleotide deletions relative to a full-length WT viral ITR
sequence while the whole wt ITR sequence is still present in the vector. For example, in a symmetric ITR such as the AAV2 ITR, if restriction sites for nicking endonuclease are each 25 bases away from the Apex, the portion after the restriction site for nicking endonuclease of the overhang does not need to be the wt IR sequence as it will be removed from the DNA
molecules after incubation with nicking endonuclease (or nicking endonuclease and restriction enzymes) and denaturing as described in Sections 5.3.3 and 5.3.4.
In certain embodiments, a hairpinned ITR sequence in the DNA molecules provide herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotide deletions relative to a full-length WT viral ITR sequence while the whole wt ITR
sequence is still present in the vector.
1003431 In some embodiments, the restriction site for nicking endonuclease is chosen based on the predicted melting temperature of the isolated nucleotide sequence present in the ITR stem region. In some embodiments, the predicted melting temperature is between 40-95 C. Further embodiments are for the restriction site for nicking endonuclease and the embodiments factoring in melting temperature are described in Sections 5.3.3, 5.3.4, 5.3.5 and 5.4.2 above.
[00344] In one embodiment, the length and GC content of the nucleotide sequence encompassing stem region of a hairpinned ITR in a DNA molecule provided herein is further modified by a deletion, insertion, and/or substitution so that a hairpin forms when the temperature is maintained at approximately 4 C. For example, the nucleotide sequence of the structural element can be modified as compared to the wild-type sequence of a viral ITR. In one embodiment, the length and GC content of the stem is designed so that a hairpin forms when the temperature is maintained at approximately 10 C or more below the melting temperature of the total ITR. The hairpin's melting temperature can be designed by changing the GC content, distance between restriction sites for nicking endonuclease and the junction closest to the primary stem (e.g. number 4 in FIG. 1), or sequence mismatch or loop, so that the melting temperature is high enough to allow the hairpinned ITR to remain folded above 50 C to ensure stable storage. The actual optimal length of the stem can vary with sequence of ITR and micro domains such as branches, loops and arms of the ITR, which can be determined according to the present disclosure in combination of the state of the art.
[00345] In some embodiments, the stem region of the hairpinned ITR encode a restriction site for Class II nicking endonuclease (e.g. NNNN downstream of 5'). In some embodiments, the stem region does not contain a restriction site for Class II
nicking endonuclease.
[00346] In some embodiments, the stem region of the hairpinned ITR encode a restriction site for Class I nicking endonuclease. In some embodiments, the stem region of the hairpinned ITR encode a restriction site for Class III, IV or V nicking endonuclease. FIG. 4 depicts various exemplary arrangements of the restriction sites for endo nuclease in the primary stem of a hairpin.
[00347] In some embodiments, the expression cassette in the hairpin-ended DNA
molecules can be any embodiments of the expression cassette described in Section 5.4,3, In certain embodiments, the ITRs in the hairpin-ended DNA molecules can be any embodiments of the IR or ITR described in Section 5.4.1. In further embodiments, the arrangement among the ITR, the expression cassette, and the restriction sites for nicking endonuclease or restriction enzymes can be any arrangement as described in Sections 5.3.3, 5.3.4, 5.3.5, 5.4.1, 5.4.2, 5.4.3 and 5.4.7.
1003481 In some embodiments, the hairpin-ended DNA comprises a top strand that is covalently linked to the 3' ITR as well as 5' ITR and once the ITR is folded, the bottom strand is flanked by two nicks (a first and a second nick) at either end of the bottom strand such that the expression cassette is in between the first nick and the second nick, wherein the first nick is formed between the 3' end of the bottom strand and the juxtaposed 5' end of the top strand as a result of top strand 5' ITR hairpin and the second nick is formed between the 5' end of the bottom strand and the juxtaposed 3' end of the top strand as a result of top strand 3' ITR hairpin.
1003491 In some embodiments, the hairpin-ended DNA comprises a bottom strand that is covalently linked to the 3' ITR as well as 5' ITR and once the ITR is folded, the top strand is flanked by two nicks (a first nick and a second nick) at either end of the top strand such that the expression cassette is in between the first nick and the second nick, wherein the first nick is formed between the 5' end of the top strand and the juxtaposed 3' end of the bottom strand as a result of bottom strand 3' ITR hairpin and the second nick is formed between the 3' end of the top strand and the juxtaposed 5' end of the bottom strand as a result of bottom strand 3' ITR hairpin.
1003501 In some embodiments, the hairpin-ended DNA comprises a top strand that is covalently linked to the 5' ITR and the bottom strand is covalently linked to the 5' ITR so that when the ITRs are folded, the first nick is formed adjacent to the bottom strand between the 3' end of the bottom strand and the juxtaposed 5' end of the top strand as a result of top strand 5' ITR hairpin and the second nick is formed adjacent to the top strand between the 3' end of the top strand and the juxtaposed 5' end of the bottom strand as a result of bottom strand 5' ITR hairpin, with the expression cassette being flanked by the first and second nicks.
1003511 In some embodiments, the hairpin-ended DNA comprises a top strand that is covalently linked to the 3' ITR and the bottom strand is covalently linked to the 3' ITR so that when the ITRs are folded, the first nick is formed adjacent to the top strand between the 5' end of the top strand and the juxtaposed 3' end of the bottom strand as a result of bottom strand 3' ITR hairpin and the second nick is formed adjacent to the bottom strand between the 5' end of the bottom strand and the juxtaposed 3' end of the top strand as a result of top strand 3' ITR hairpin, with the expression cassette being flanked by the first and second nicks.
1003521 In some embodiments, the hairpin-ended DNA comprising the two nicks as described in this Section (Section 5.5) and the preceding 4 paragraphs can be ligated to repair the nicks by forming a covalent bond between the two nucleotides flanking the nick. In some embodiments, one of the two nicks described in this Section (Section 5.5) and the preceding 4 paragraphs can be ligated and repaired such that when denatured, the DNA
molecule becomes a linear single stranded DNA molecule. In some embodiments, the two nicks described in this Section (Section 5.5) and the preceding 4 paragraphs can be ligated and repaired such that when denatured, the DNA molecule becomes a circular single stranded DNA molecule.
1003531 In some embodiments, the two flanking ITR pairs in the hairpin-ended DNA
molecule comprise identical DNA sequence In some embodiments, the two flanking ITR
pairs in the hairpin-ended DNA molecule comprise different DNA sequences. In some embodiments, one of the ITRs in the hairpin-ended DNA molecule is modified by deletion, insertion, and/or substitution as compared to the other ITR in the same hairpin-ended DNA
molecule. In another embodiment, the first ITR and the second ITR in the hairpin-ended DNA molecule are both modified, e.g. by deletion, insertion, and/or substitution. In yet another embodiment, the first ITR and the second ITR in the hairpin-ended DNA
molecule comprise different DNA sequences and are both modified. In a further embodiment, the first ITR and the second ITR in the hairpin-ended DNA molecule comprise different DNA
sequences and are both modified, wherein the modifications for the two ITRs are different.
In yet a further embodiment, the first ITR and the second ITR in the hairpin-ended DNA
molecule comprise different DNA sequences and are both modified, wherein the modifications for the two ITRs are identical. In one embodiment, the first ITR
and the second ITR in the hairpin-ended DNA molecule comprise identical DNA sequence and are both modified, wherein the modifications for the two ITRs are different. In one embodiment, the first ITR and the second ITR in the hairpin-ended DNA molecule comprise identical DNA sequence and are both modified, wherein the modifications for the two ITRs are identical. In one embodiment, the first ITR and the second ITR in the hairpin-ended DNA
are both modified ITRs and the two modified ITRs are not identical. In some embodiments, the hairpin-ended DNA molecules comprise two ITRs that are asymmetric, wherein the asymmetry can be a result of any changes in one ITR that are not reflected in the other ITR.
In certain embodiments, the hairpin-ended DNA molecules comprise two ITRs that are asymmetric, wherein the ITRs are different with respect to each other in any way. In certain embodiments, the modifications provided in this paragraph, including deletion, insertion, and/or substitution, can be any such modifications described above in this Section (Section 5.5).
1003541 In one aspect, a hairpin-ended DNA molecule provided herein comprises, in the 5' to 3' direction: a first IR, a nucleotide sequence of interest and a second IR. In one embodiment, the nucleotide sequence of interest comprises an expression cassette as described herein, e.g. in Sections 5.4.3. In certain embodiments, the hairpin-ended DNA
molecules provided herein including in Section 3 and this Section (Section 5.5) comprise an expression cassette, wherein the expression cassette can be any embodiments described in Sections 3 and 5.4.3.
1003551 The hairpin-ended DNA molecules can comprise a combination of dsDNA
and ssDNA In some embodiments, certain portion of the hairpin-ended DNA molecules provided in this Section (Section 5.5) is dsDNA. In further embodiments, the dsDNA portion of the hairpin-ended DNA molecules provided in this Section (Section 5.5) comprises the expression cassette, a stem region of the ITR, or both. In one embodiment, the dsDNA
portion of the hairpin-ended DNA molecules provided in this Section (Section 5.5) accounts for over 90% of the hairpin-ended DNA molecules. In another embodiment, the dsDNA
portion of the hairpin-ended DNA molecules provided in this Section (Section 5.5) accounts for at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the hairpin-ended DNA molecules. In another embodiment, the dsDNA portion of the hairpin-ended DNA molecules provided in this Section (Section 5.5) accounts for about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the hairpin-ended DNA
molecules.
1003561 In some embodiments, the hairpin-ended DNA molecule provided herein can be efficiently targeted or transported to the nucleus of a cell. In one embodiment, the hairpin-ended DNA molecule provided herein can be efficiently targeted or transported to the nucleus of a cell by the binding between the aptamer formed at the ITR and a nucleus protein. In another embodiment, the hairpin-ended DNA molecule provided herein can be efficiently targeted or transported to the nucleus of a cell, such that the abundance of the hairpin-ended DNA molecules in the nucleus is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that in the cytoplasm. In yet another embodiment, the hairpin-ended DNA
molecule provided herein can be efficiently targeted or transported to the nucleus of a cell, such that the abundance of the hairpin-ended DNA molecules in the nucleus is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 fold higher than that in the cytoplasm.
1003571 In various embodiments of the hairpin-ended DNA molecule provided herein including in Section (Section 5.5), the hairpin-ended DNA molecule lacks the RABS and/or TRS sequences as described in Section 5.4.5. In others embodiments of the hairpin-ended DNA molecule provided herein including in Section (Section 5.5), the hairpin-ended DNA
molecule lacks any or any combination of the DNA sequences, elements, or features as described in Section 5.4.5.
1003581 In some additional embodiments, embodiments of the hairpin-ended DNA
molecule provided herein including in Section (Section 55), the hairpin-ended DNA
molecule can be an isolated hairpin-ended DNA molecules in any embodiment with respect to purity as described in Section 5.4.8.
5.6 Functional Properties of the Hairpin-ended DNA Molecules 1003591 In some embodiments, the ITR promotes the long-term survival of the nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITR promotes the permanent survival of the nucleic acid molecule in the nucleus of a cell (e.g., for the entire life-span of the cell). In some embodiments, the ITR promotes the stability of the nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITR inhibits or prevents the degradation of the nucleic acid molecule in the nucleus of a cell.
1003601 In some embodiments, when the ITR assumes its folded state, it is resistant to exonuclease digestion (e.g. exonuclease V), e.g. for over an hour at 37 C. In one embodiment, the hairpin-ended DNA molecule is resistant to exonuclease digestion (e.g.
digestion by exonuclease V). In another embodiment, the hairpin-ended DNA
molecule is resistant to exonuclease digestion (e.g. digestion by exonuclease V) for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more hours In yet another embodiment, the hairpin-ended DNA molecule is resistant to exonuclease digestion (e.g. digestion by exonuclease V) for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 hours.
1003611 As unexpectedly found by the inventors and provided herein, duplex linear DNA
vectors with ITRs similar to viral ITRs can be produced without the need for Rep proteins and consequently independent of the RABS or TRS sequence for genome replication.
Accordingly, the RBE and TRS can optionally be encoded in the nucleotide sequence disclosed herein but are not required and offer flexibility with regard to designing the ITRs.
In one embodiment, the DNA molecules provided herein comprise ITRs that do not comprise RABS. In another embodiment, the DNA molecules provided herein comprise ITRs that do not comprise TRS. In yet another embodiment, the DNA molecules provided herein comprise ITRs that do not comprise either RABS or TRS. In a further embodiment, the DNA
molecules provided herein comprise ITRs that comprise RABS, TRS, or both RABS
and TRS.
1003621 In some embodiments, the hairpin-ended DNA molecules provided herein are stable in the host cell. In some embodiments, the hairpin-ended DNA molecules provided herein are stable in the host cell for long term culture.
1003631 In certain embodiments, the hairpin-ended DNA molecules provided herein can be efficiently delivered to a host cell.
1003641 The DNA molecules provided herein have superior stability not just for their resistance to exonuclease digestion described above, but also with respect to their structure.
In one embodiment, the structure of the DNA molecules remains the same after storage at room temperature for 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In another embodiment, the ensemble structure of the DNA molecules remains the same after storage at room temperature for 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the structure of the DNA molecules provided herein is the same after 2, 3, 4, 5, or 20 cycles of denaturing/renaturing (e.g. denaturing as described in Section 5.3.3 and re-annealing as described in Section 5.3.5). . DNA structures can be described by an ensemble of structures at or around the energy minimum. In certain embodiments, the ensemble DNA
structure is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing. In one embodiment, the folded hairpin structure formed from the ITR or IR provided herein is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing. In another embodiment, the ensemble structure of the folded hairpin is the same after 2, 3, 4, 5, 10 or 20 cycles of denaturing/renaturing.

5.7 Delivery Vehicles Comprising the Hairpin-ended DNA
Molecules 1003651 In some embodiments, the hairpin-ended DNA molecules provided herein can be delivered via a hydridosome as described in USPN 10,561,610, which is herein incorporated in its entirety by reference. In other embodiments, the DNA molecules provided herein can be delivered via a hydridosome.
1003661 In certain embodiments, the DNA molecules provided herein can be delivered via lipid particles including lipid nanoparticles. In other embodiments, the hairpin-ended DNA
molecules provided herein can be delivered via lipid nanoparticles. In some embodiments, the lipid nanoparticle comprises any one or more lipids selected from ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEGylated lipid. In one embodiment, the lipid particle comprises any one or more lipids selected from ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEGylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent or 40 to 60 mole percent for the ionizable lipid, the mole percent of non-cationic lipid ranges from 0 to 30 or 0 to 15, the mole percent of sterol ranges from 20 to 70 or 30 to 50, and the mole percent of PEGylated lipid ranges from 1 to 6 or 2 to 5. In another embodiment, the lipid particle comprises any one or more lipids selected from ionizable lipid, non-cationic lipid (e.g.
phospholipid), a sterol (e.g., cholesterol) and a PEGylated lipid, where the molar ratio of lipids ranges from 40 to 60 mole percent for the ionizable lipid, the mole percent of non-cationic lipid ranges from 0 to 15, the mole percent of sterol ranges from 30 to 50, and the mole percent of PEGylated lipid ranges from 2 to 5. In yet another embodiment, the lipid particle comprises any one or more lipids selected from ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEGylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, the mole percent of non-cationic lipid ranges from 0 to 30, the mole percent of sterol ranges from 20 to 70, and the mole percent of PEGylated lipid ranges from 1 to 6.
1003671 The disclosure provides that ionizable lipids can be used employed to condense the nucleic acid cargo, at low pH and to drive membrane association and fusogenicity. Such ionizable lipids can be used as part of the delivery vehicle for the compositions of and methods for the DNA molecules provided herein. In some embodiments, ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower. In some embodiments, ionizable lipids have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, for example at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form.
Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.
1003681 Further exemplary ionizable lipids are described in PCT patent publications W02015/095340, W02015/199952, W02018/011633, W02017/049245, W02015/061467, W02012/040184, W02012/000104, W02015/074085, W02016/081029, W02017/004143, W02017/075531, W02017/117528, W02011/022460, W02013/148541, W02013/116126, W02011/153120, W02012/044638, W02012/054365, W02011/090965, W02013/016058, W02012/162210, W02008/042973, W02010/129709, W02010/144740 , W02012/099755, W02013/049328, W02013/086322, W02013/086373, W02011/071860, W02009/132131, W02010/048536, W02010/088537, W02010/054401, W02010/054406 , W02010/054405, W02010/054384, W02012/016184, W02009/086558, W02010/042877, W02011/000106, W02011/000107, W02005/120152, W02011/141705, W02013/126803, W02006/007712, W02011/038160, W02005/121348, W02011/066651, W02009/127060, W02011/141704, W02006/069782, W02012/031043, W02013/006825, W02013/033563, W02013/089151, W02017/099823, W02015/095346, and W02013/086354, all of which are herein incorporated in their entirety by reference.
1003691 In some specific embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 1Z)-heptatriaconta-6,9,28,3 1-tetraen-19-y1-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3).
1003701 In some embodiments, the lipid nanoparticles encapsulation the DNA
molecule of provided herein include one or more lipids selected from the group consisting of distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), dipalmitoyl-phosphatidylcholine (DPPC), dioleoyl-phosphatidylglycerol (DOPG), dipalmitoyl-phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidy-lethanolamine, dipalmitoyl-phosphatidyl-ethanolamine (DPPE), dimyristoylphospho-ethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, 1-stearioy1-2-oleoyl-phosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
[00371] Delivery vehicles provided herein include those for delivering the DNA
molecules provided herein to cells, which sometime are referred to as transfection.
Further useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation.
Transfection reagents well known in the art are provided herein and include, but are not limited to, TurboFeet Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASSTm P Protein Transfection Reagent (New England Biolabs), CHARIOTTm Protein Delivery Reagent (Active Motif), PROTE0JUICET1I Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECT AMINETm 2000, LIPOFECT
AIVIIINETM 3000 (Thermo Fisher Scientific), LIPOFECT AIVIINETM (Thermo Fisher Scientific), LIPOFECTINTm (Thermo Fisher Scientific), DMRIE-C, CELLFECTINTm (Thermo Fisher Scientific), OLIGOFECT AMINETm (Thermo Fisher Scientific), LIPOFECT
ACETM, FUGENETM (Roche, Basel, Switzerland), FUGENETM HD (Roche), TRANSFECT
AMTm(Transfectam, Promega, Madison, Wis.), TFX-10Tm (Promega), TFX-20Tm (Promega), TFX-50Tm (Promega), TRANSFECTINTm (BioRad, Hercules, Calif.), SILENTFECTTm (Bio-Rad), EffecteneTM (Qiagen, Valencia, Calif.), DC-chol (Avanti Polar Lipids), GENEPORTERTm (Gene Therapy Systems, San Diego, Calif), DHARMAFECT 1TM
(Dharmacon, Lafayette, Colo.), DHARMAFECT 2TM (Dharmacon), DHARMAFECT 3TM
(Dharmacon), DHARMAFECT 4TM (Dharmacon), ESCORTTm III (Sigma, St. Louis, Mo.), and ESCORTTm IV (Sigma Chemical Co.) [00372] In some cases, chemical delivery systems can be used to deliver the DNA
molecules provided herein, for example, by using cationic transfection reagents, which include compaction of negatively charged nucleic acid by polycationic chemicals to form cationic liposome/micelle or cationic polymers Cationic lipids used for the delivery method include, but not limited to monovalent cationic lipids, polyvalent cationic lipids, guanidine containing compounds, cholesterol derivative compounds, cationic polymers, (e.g., poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.
[00373] In some embodiments, DNA molecules provided herein are delivered by making transient penetration in cell membrane by applying mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance into the targeted cells is facilitated. For example, a DNA molecule provided herein can be delivered by transiently disrupting cell membrane by squeezing the cell through a size-restricted channel or by other means known in the art.
1003741 The disclosure provides that the DNA molecules provided herein can be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.
1003751 Relative amounts of the active ingredient (e.g. DNA molecules provided herein or cells comprising DNA molecules provided herein for transfer or transplantation into a subject), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 01% and 99% (w/w) of the active ingredient By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
1003761 Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, exosomes, extracellular vesicles, hybridosomes polymers, peptides, proteins, cells comprising DNA molecules provided herein (e.g., for transfer or transplantation into a subject) and combinations thereof.
1003771 In the case of viral particles, exosomes or hybridosomes, which may contain endogenous nucleic acids, quantification of DNA molecules may be used as the measure of the dose contained in the formulation. Any method known in the art can be used to determine the DNA molecules number of the compositions of the disclosure. One method for performing DNA molecule number titration is as follows: samples of viral particles, exosomes or hybridosomes compositions comprising hairpin-ended DNA encoding GDE are first treated with DNase to eliminate contaminating host DNA from the production process.
The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (for example poly A
signal). Another suitable method for determining genome copies is the quantitative- PCR (qPCR), particularly the optimized qPCR or digital droplet PCR.
1003781 Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term "pharmaceutical composition" refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
1003791 In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
As used herein, the phrase "active ingredient" generally refers to either DNA molecules provided herein or cells or substance comprising the DNA molecules provided herein.
1003801 Formulations of the DNA molecules and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit 1003811 In some embodiments, the formulations described herein may contain sufficient DNA molecules or active ingredients for expression of the ORFs in the expression cassette for the treatment of a disease.
1003821 In some embodiments, DNA molecules of the present disclosure are substantially free of any viral proteins such as AAV Rep78. In some embodiments, the isolated DNA
molecules of the disclosure are 100% free, 99% free, 98% free, 97% free, 96%
free, 95%
free, 94% free, 93% free, 92% free, 91% free, or 90% free of viral proteins.
1003831 The DNA molecules of the present disclosure can be formulated using one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the active ingredients; (4) alter the biodistribution (e.g., target the DNA molecules or active ingredients comprising the DNA
molecules to specific tissues or cell types); (5) increase the translation of ORFs in the expression cassette; (6) alter the release profile of the protein encoded by the ORFs of the expression cassette and/or (7) allow for regulatable expression of the ORFs of the expression cassette.
1003841 In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration.
In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
1003851 Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R.
Gennaro, Lippincott, Williams & Wilkins, Baltimore, 1VID, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
1003861 Exemplary diluents include those known and used in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams &
Wilkins, Baltimore, MD, 2006.) 1003871 In some embodiments, the pharmaceutical composition for the DNA
molecules provided herein can comprise at least one inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations.
In some embodiments, all, none or some of the inactive ingredients used in the formulations of the present disclosure can be any one of such approved by the US Food and Drug Administration (FDA) and used in the art.
5.8 Method of Using 1003881 The disclosure provides that the DNA molecules provided herein can be used to deliver the ORFs or transgenes in the expression cassette to a cell for expression. ORFs or transgenes as described in Section 5.4.3 can be efficiently delivered. The disclosure provides that the DNA molecules provided herein can be used to deliver the ORFs or transgenes in the expression cassette to a human subject. Any ORFs or transgenes as described in Section 5.4.3 can be efficiently delivered.
1003891 In one specific embodiment, the method of delivering a gene of interest to a cell for expression comprises: transfecting the DNA molecules provided herein into the cell.

In certain embodiments, the cell is a human cell. In another embodiment, the cell is a human primary cell. In yet another embodiment, the cell is a primary human blood cell. In one embodiment, the DNA molecules can be transfected into the cell via any delivery vehicles described in Section 5.7.
1003901 In another specific embodiment, the method of delivering a gene of interest to a human subject for expression comprises: transfecting the DNA molecules provided herein into a cell and administering the cell to a human subject. In certain embodiments, the cell is a human cell. In another embodiment, the cell is a human primary cell. In yet another embodiment, the cell is a primary human blood cell. In one embodiment, the DNA
molecules can be transfected into the cell via any delivery vehicles described in Section 5.7.
1003911 In some embodiments, the DNA molecules provided herein can be used in gene therapy by delivering a disease correcting genes in the expression cassette into a cell or a human subject as described in the preceding 3 paragraphs 1003921 In certain embodiments, the DNA molecules provided herein can be used to transfect cells that are difficult to transfect as known in the art. Such cells known to be difficult to transfect include cells that are not actively dividing. In some embodiments, such cells can be human primary cells, including, for example, human primary blood cells, human primary hepatocyte, human primary neurons, human primary muscle cells, human primary cardiomyocyte, etc.
5.8.1 Host cell As used herein, the term "host cell", includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or hairpin ended expression vector of the present disclosure.
1003941 In some embodiments, a hairpin ended vector for expression of GDE
protein as disclosed herein delivers the GDE protein transgene into a subject host cell.
In some embodiments, the subject host cell is a human host cell, including, for example blood cells, stem cells, hematopoietic cells, CD34-h cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cell of mammalian origin, including, without limitation, hepatic (i.e., liver) cells, lung cells, cardiac cells, pancreatic cells, intestinal cells, diaphragmatic cells, renal (i.e., kidney) cells, neural cells, blood cells, bone marrow cells, or any one or more selected tissues of a subject for which gene therapy is contemplated. In one aspect, the subject host cell is a human host cell.

1003951 The present disclosure also relates to recombinant host cells as mentioned above, including a hairpin ended vector for expression of GDE protein as disclosed herein. Thus, one can use multiple host cells depending on the purpose as is obvious to the skilled artisan. A
hairpin ended vector for expression of GDE protein as disclosed herein can be introduced into a host cell so that the donor sequence is maintained as a chromosomal integrant. The term host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the donor sequence and its source.
1003961 The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. In one embodiment, the host cell is a human cell (e.g., a primary cell, a stem cell, or an immortalized cell line). In some embodiments, the host cell can be administered a hairpin ended vector for expression of GDE protein as disclosed herein ex vivo and then delivered to the subject after the gene therapy event A host cell can be any cell type, e g , a somatic cell or a stem cell, an induced pluripotent stem cell, or a blood cell, e.g., T-cell or B-cell, or bone marrow cell. In certain embodiments, the host cell is an allogenic cell. In some embodiments, gene modified host cells, e.g., bone marrow stem cells, e.g., CD34+ cells, or induced pluripotent stem cells can be transplanted back into a patient for expression of a therapeutic protein.
1003971 GDE is predominantly expressed in the liver, heart, skeletal muscles and thyroid.
During fetal development, GDE can be expressed in the adrenal gland, heart, intestine, kidney lung, and stomach. Accordingly, one can administer a hairpin ended vector expressing GDE to any one or more tissues selected from: liver, kidneys, gallbladder, prostate, adrenal.
In some embodiments, when a hairpin ended vector expressing GDE is administered to an infant, or administered to a subject in utero, one can administer a hairpin ended vector expressing GDE to any one or more tissues selected from: liver, skeletal muscle, heart, tongue, lung, and stomach.
1003981 In some embodiments, a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein can be used to deliver an GDE protein to skeletal, cardiac or diaphragm muscle, for production of an GDE protein for secretion and circulation in the blood or for systemic delivery to other tissues to treat, ameliorate, and/or prevent GSDIII.
1003991 In other embodiments herein, the term host cell refers to cultures of liver or muscle cells of various mammalian species for in vitro assessment of the compositions described herein. Still in other embodiments, the term "host cell" is intended to reference the liver cells or muscle of the subject being treated in vivo for GSDIII disease.

5.8.2 Testing for successful gene expression using a hairpin-ended DNA
molecule 1004001 Assays well known in the art can be used to test the efficiency of gene delivery of an GDE protein by a hairpin-ended DNA molecule can be performed in both in vitro and in vivo models. Levels of the expression of the GDE protein by the hairpin-ended DNA can be assessed by one skilled in the art by measuring mRNA and protein levels of the GDE protein (e.g., reverse transcription PCR, western blot analysis, and enzyme-linked immunosorbent assay (ELISA)). In one embodiment, the DNA comprises a reporter protein that can be used to assess the expression of the GDE protein, for example by examining the expression of the reporter protein by fluorescence microscopy or a luminescence plate reader.
For in vivo applications, protein function assays can be used to test the functionality of a given GDE
protein to determine if gene expression has successfully occurred. One skilled will be able to determine the best test for measuring functionality of an GDE protein expressed by the hairpin-ended DNA molecule in vitro or in vivo.
1004011 It is contemplated herein that the effects of gene expression of an GDE protein from the DNA vector in a cell or subject can last for at least 0.5 month, at least 1 month, at least 2 months, at least 3 months, at least four months, at least 5 months, at least six months, at least 10 months, at least 12 months, at least 18 months, at least 2 years, at least 5 years, at least 10 years, at least 20 years, or can be permanent.
1004021 In some embodiments, an GDE protein in the expression cassette, expression construct, or hairpin-ended DNA molecule described herein can be codon optimized for the host cell. As used herein, the term "codon optimized" or "codon optimization"
refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human (e.g., humanized), by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular bias for certain codons of a particular amino acid. Typically, codon optimization does not alter the amino acid sequence of the original translated protein Optimized codons can be determined using e.g., Aptagen's Gene Forge codon optimization and custom gene synthesis platform (Aptagen, Inc.) or another publicly available database.

5.9 Methods of Treatment 1004031 In another aspect, provided herein are methods for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a patient, the method comprising administering to the patient a DNA molecule comprising a transgene encoding human GDE or a catalytically active fragment thereof. In specific embodiments, the DNA molecule is contained in a hybridosome. In a specific embodiment, the DNA molecule is contained in a lipid nanoparticle.
1004041 The DNA molecular may be contained in a single vector or in multiple vectors which are co-administered.
1004051 In some embodiments, the patient treated in accordance with the methods described herein is an adult. In some embodiments, the patient is a pediatric patient. The pediatric patient may be, for example, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, or about 18 years old. In some embodiments, the pediatric patient is an infant. As used herein, the terms "patient- and "subject- are used interchangeably. In some embodiments, the patient is human.
1004061 In specific embodiments, the disease treated in accordance with the methods described herein is Glycogen Storage Disease (GSD) Type III (GSDIII). In specific embodiments, the disease is GSDIIIa, GSDIIIb, GSDIIIc, or GSDIIId.
1004071 In specific embodiments, a method of treatment described herein further comprises administering one or more additional therapies to the patient. The one or more additional therapy may be administered prior to, concurrently with, or subsequently to the DNA molecule described herein. In specific embodiments, the additional therapy is for the treatment of a disease associated with reduced activity of GDE. In specific embodiments, the additional therapy is immunosuppressive therapy. In specific embodiments, a patient treated in accordance with the methods described herein is does not receive immunosuppressive therapy.
5.9.1 Determining Efficacy by Assessing GDE protein Expression from the DNA vector 1004081 Essentially any method known in the art for determining protein expression can be used to analyze expression of a GDE protein from a hairpin-ended DNA molecule.
Non-limiting examples of such methods/assays include enzyme-linked immunoassay (ELISA), affinity ELISA, ELISPOT, serial dilution, flow cytometry, surface plasmon resonance analysis, kinetic exclusion assay, mass spectrometry, Western blot, immunoprecipitation, and PCR.
1004091 For assessing GDE protein expression in vivo, a biological sample can be obtained from a subject for analysis. Exemplary biological samples include a biofluid sample, a body fluid sample, blood (including whole blood), serum, plasma, urine, saliva, a biopsy and/or tissue sample etc. A biological sample or tissue sample can also refer to a sample of tissue or fluid isolated from an individual including, but not limited to, tumor biopsy, stool, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, breast milk, cells (including, but not limited to, blood cells), tumors, organs, and also samples of in vitro cell culture constituent. The term also includes a mixture of the above-mentioned samples.
The term "sample" also includes untreated or pretreated (or pre-processed) biological samples In some embodiments, the sample used for the assays and methods described herein comprises a serum sample collected from a subject to be tested.
5.9.2 Determining Efficacy of the expressed GDE protein by Clinical Parameters 1004101 The efficacy of a given GDE protein expressed by a hairpin-ended DNA
molecule for GSDIII (i.e., functional expression) can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of GSDIII is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10%
following treatment with a DNA vector described herein, encoding a therapeutic GDE protein as described herein. Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of GSDIII, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting GSDIII, e.g., arresting, or slowing progression of GSDIII; or (2) relieving the GSDIII, e.g., causing regression of GSDIII symptoms; and (3) preventing or reducing the likelihood of the development of the GSDIII disease, or preventing secondary diseases/disorders associated with GSDIII. An effective amount for the treatment of a disease means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators that are particular to GSDIII
disease. A physician can assess for any one or more of clinical symptoms of GSDIII which include: severe fasting intolerance, growth failure, and hepatomegaly.
Furthermore, biochemical characteristics are (non)ketotic hypoglycemia, hyperlactatemia, increased liver enzymes, and hyperlipidemia. Routine analysis in plasma (i.e., glucose, lactate, ketones, alanine and aspartate aminotransferases [ALT and AST], creatine phosphokinase [CK], uric acid, lipids) and urine (ketones) are essential for monitoring metabolic control. Methods and reference values for plasma analysis and metabolic monitoring have been described in the art (e.g. Touati G., Mochel F., Rabier D. (2012) Diagnostic Procedures: Functional Tests and Post-mortem Protocol. In: Saudubray TM., van den Berghe G., Walter J.H. (eds) Inborn Metabolic Diseases. Springer, Berlin, Heidelberg) Specifically reduced urinary glucose tetrasaccharide (Glc4), a metabolite resulting from enzymatic degradation of glycogen by amylase, on a regular diet. Monitoring urinary Glc4 as well as urine hexose tetrasaccharide (Hex4) may represent a biomarker in the development of treatments for GSDIII.
Urinary Glc4 concentration can be determined by stable isotope-dilution electrospray tandem mass spectrometry as previously described (Young, S.P. et al. (2003) Biochem, 316(2): 175-80).
1004111 In some embodiments, a method of treatment described herein results in a reduction in the number of events during which blood lactate levels are above 2 mmol/L, above 3mmo1/L, or above 4 mmol/L for 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-11 hours, and 11-12 hours in a subject.
1004121 In some embodiments, a method of treatment described herein results in a reduction in hyperlipidemic episodes in a subject. By "hyperlipidemic episode"
is meant an increase in total blood cholesterol to above 200 mg/dL and/or an increase in blood triglycerides to above 150 mg/dL for 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-11 hours, and 11-12 hours.
1004131 In one embodiment, a physician can further assess the efficacy of the expressed GDE protein for any one or more of metabolism related clinical symptoms of GSDIII
including glycemia. Specifically, efficacy of expressed GDE can be assessed by monitoring the ability maintain normoglycemia or the prevention of hypoglycemia during fasting, or in absence of frequent meals enriched in complex carbohydrates, administration of uncooked cornstarch and/or, depending on age of the patient and fasting tolerance, overnight continuous enteral feeding. In one embodiment, the efficacy of the expressed GDE proteins can be partial restoration of the normoglycemic status after lh, 2h, 3h, 4h, 6h, 8h, or 9h after the last meal of the patient.
1004141 In one embodiment, a physician can further assess the efficacy of the expressed GDE protein for any one or more of metabolism related clinical symptoms of GSDIII
including glycemia. Specifically, efficacy of expressed GDE can be assessed by monitoring the ability maintain normoglycemia or the prevention of hypoglycemia during fasting, or in absence of frequent meals enriched in complex carbohydrates, administration of uncooked cornstarch and/or, depending on age of the patient and fasting tolerance, overnight continuous enteral feeding. In one embodiment, the efficacy of the expressed GDE proteins can be partial restoration of the normoglycemic status within lh, 2h, 3h, 4h, 6h, 8h, or 9h after the last meal of the patient.
1004151 In one aspect, a coding sequence is provided which encodes a functional GDE
protein By "functional GDE", is meant a gene which encodes an GDE protein which provides at least about 50%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of the native GDE
protein, or a natural variant or polymorph thereof which is not associated with disease. A
variety of assays exist for measuring GDE expression and activity levels in vitro. (see Maire et al, (1991), Clinical Biochemistry, 24(2), 169-178, and DiMauro et al, Pediatr Res. 1973 7(9):739-44.) 1004161 In some embodiments the hairpin-ended DNA molecules encoding a functional GDE protein can be delivered to the liver, in particular to hepatocytes, of a patient in need (e.g. , a GSDIII patient), and can elevate active GDE levels of the patient.
The hairpin-ended DNA molecule can be used for preventing, treating, ameliorating or reversing any symptoms of GSDIII in the patient.

In further aspects, a hairpin-ended DNA molecule of this disclosure can also be used for reducing the dependence of a GSDIII patient on a particular diet to control the disease. For instance, a hairpin-ended DNA molecule of this invention can be used to reduce a GSDIII patient's dependence on frequent high carbohydrate meals and/or diets abnormally high in protein.
1004181 In other exemplary embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of limit dextrin levels in a biological sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of limit dextrin accumulation in a biological sample (e.g. , a liver sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%
as compared to baseline limit dextrin levels before treatment. In some embodiments, the biological sample is a portion of an organ selected from liver, heart, diaphragm, quadriceps, and gastrocnemius. In an exemplary embodiment, the biological sample is a liver section, e.g., a section of hepatocytes. In a further exemplary embodiment, a therapeutically effective dose, when administered regularly, results in at least a 50%, 60%, 70%, or 80% reduction of limit dextrin levels in a liver sample as compared to baseline limit dextrin levels before treatment.
5.9.3 Administration 1004191 A DNA molecule described herein may be administered to a subject once or repeatedly Thus, in specific embodiments, a method for treating a disease associated with reduced activity of GDE in a human patient comprises the steps of (i) administering a first dose of a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof to the patient and (ii) administering a second dose of the DNA molecule to the patient.
1004201 In some embodiments, the first dose of the DNA molecule is administered to the patient at least one month, at least two months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months before the second dose of the DNA molecule. In some embodiments, the first dose of the DNA molecule is administered to the patient at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, or at least 20 years before the second dose of the DNA molecule.
1004211 In some embodiments, the first dose of the DNA molecule is administered about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-15 months, about 15-18 months, about 18-21 months, about 21-24 months, about 24-27 months, about 27-30 months, about 30-33 months, about 33-36 months, about 3-4 years, about 4-5 years, about 5-6 years, about 6-7 years, about 8-9 years, about 9-10 yeasts, about 1 0-1 1 years, about 11-12 years, about 12-13 years, about 13-14 years, about 14-15 years, about 15-16 years, about 16-17 years, about 17-18 years, about 18-19 years, or about 19-20 years before the second dose of the DNA molecule.

1004221 The first dose of the double-stranded DNA molecule and the second dose of the DNA molecule may contain the same amount of the DNA molecule or different amounts of the DNA molecule.
1004231 In some embodiments, a method of treatment described herein further comprises administering one or more additional doses of the DNA molecule, e.g., administering a total of 3, 4, 5, 6,7 8, 9, or 10 doses of the DNA molecule.
1004241 The DNA molecule may be administered once weekly, biweekly (every other week), or monthly. In some embodiments, the DNA molecule is administered about every 3 months, about every 6 months, about every 9 months, about every 12 months, about every 15 months, about every 18 months, about every 21 months, about every 2 years, about every 3 years, about every 4 years, about every 5 years, about every 6 years, about every 7 years, about every 8 years, about every 9 years, about every 10 years, about every 11 years, about every 12 years, about every 13 years, about every 14 years, about every 15 years, about every 16 years, about every 17 years, about every 18 years, about every 19 years, or about every 20 years.
1004251 In specific embodiments, the DNA molecule is administered to the patient for the duration of the life of the patient.
1004261 A DNA molecule described herein may be administered to a subject by any suitable route. In certain embodiments, said route of administration is selected from the group consisting of intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. In a specific embodiment, said route is intravenous. In other embodiments, said route is an administration route delivering the hairpin-ended DNA to the liver that is other than intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal.
1004271 In some embodiments, a method of treating a disease in a subject comprises introducing into a target cell in need thereof (in particular a muscle cell or tissue) of the subject, a therapeutically effective amount of a hairpin ended molecule encoding a GDE
protein, optionally with a pharmaceutically acceptable carrier. In some embodiments, the hairpin-ended DNA molecule for expression of GDE protein, is administered to a muscle tissue of a subject.
1004281 In some embodiments, administration of the hairpin-ended DNA molecule can be to any site in a subject, including, without limitation, a site selected from the group consisting of a smooth muscle, skeletal muscleõ the heart, the diaphragm, or muscles of the eye.

1004291 Administration of a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein, to a skeletal muscle according to the present disclosure includes but is not limited to administration to the skeletal muscle in the limbs (e.g., upper leg, lower leg, upper arm and/or lower arm), thorax, abdomen, back, neck, head (e.g., tongue), pelvis/perineum, and/or digits.. The hairpin-ended DNA molecule as disclosed herein can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion, (optionally, isolated limb perfusion of a leg and/or arm; see, e.g. Arruda et al., (2005) Blood 105: 3458-3464), and/or direct intramuscular injection. In particular embodiments, the hairpin-ended DNA molecule encoding GDE as disclosed herein is administered to the liver, eye, a limb (e.g., arm and/or leg) of a subject (e.g., a subject with GSDITI) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-articular administration.
1004301 Furthermore, a composition comprising a hairpin-ended DNA molecule for expression of GDE protein, as disclosed herein, which is administered to a skeletal muscle, can be administered to a skeletal muscle in the limbs (e.g., upper leg, lower leg, upper arm and or lower arm,), thorax, abdomen, back, neck, head (e.g., tongue), pelvis/perineum, and/or digits. Suitable skeletal muscles include but are not limited to abductor digiti minimi (in the hand), abductor digiti minimi (in the foot), abductor hallucis, abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articularis genus, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, digastric, dorsal interossei (in the hand), dorsal interossei (in the foot), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimi brevis (in the foot), flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis, iliocostalis lumborum, iliocostalis thoracis, illiacus, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, inter spinalis, intertransversi, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator labii superioris alaeque nasi, levator palpebrae superioris, levator scapulae, long rotators, longissimus capitis, longissimus cervicis, longissimus thoracis, longus capitis, longus colli, lumbricals (in the hand), lumbricals (in the foot), masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, obliquus capitis inferior, obliquus capitis superior, obturator externus, obturator intemus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interossei, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis, plantar interossei, plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantae, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, sartorius, scalenus minimus, semimembranosus, semispinalis capitis, semispinalis cervicis, semispinalis thoracis, semitendinosus, serratus anterior, short rotators, soleus, spinalis capitis, spinalis cervicis, spinalis thoracis, splenius capitis, splenius cervicis, sternocleidomastoid, stemohyoid, stemothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor, and any other suitable skeletal muscle as known in the art.
1004311 In certain embodiments Administration of a hairpin-ended DNA molecule for the expression of GDE protein, as disclosed herein, to diaphragm muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration.
1004321 Administration of a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum The hairpin-ended DNA molecule as described herein can be delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion 1004331 Administration of a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein to smooth muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration. In one embodiment, administration can be to endothelial cells present in, near, and/or on smooth muscle. Non-limiting examples of smooth muscles include the iris of the eye, bronchioles of the lung, laryngeal muscles (vocal cords), muscular layers of the stomach, esophagus, small and large intestine of the gastrointestinal tract, ureter, detrusor muscle of the urinary bladder, uterine myometrium, penis, or prostate gland.
1004341 In some embodiments, a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle. In representative embodiments, a hairpin-ended DNA molecule according to the present disclosure is used to treat and/or prevent disorders of skeletal, cardiac and/or diaphragm muscle.
1004351 In some embodiments a composition comprising a hairpin-ended DNA
molecule for expression of GDE protein as disclosed herein, can be delivered to one or more muscles of the eye (e.g., Lateral rectus, Medial rectus, Superior rectus, Inferior rectus, Superior oblique, Inferior oblique), facial muscles (e.g., Occipitofrontalis muscle, Temporoparietalis muscle, Procerus muscle, Nasalis muscle, Depressor septi nasi muscle, Orbicularis oculi muscle, Corrugator supercilii muscle, Depressor supercilii muscle, Auricular muscles, Orbicularis oris muscle, Depressor anguli oris muscle, Risorius, Zygomaticus major muscle, Zygomaticus minor muscle, Levator labii superioris, Levator labii superioris alaeque nasi muscle, Depressor labii inferioris muscle, Levator anguli oris, Buccinator muscle, Mentalis) or tongue muscles (e.g., genioglossus, hyoglossus, chondroglossus, styloglossus, palatoglossus, superior longitudinal muscle, inferior longitudinal muscle, the vertical muscle, and the transverse muscle).
1004361 In some embodiments, a composition comprising a hairpin-ended DNA
molecule for expression of GDE protein, as disclosed herein, can be injected into one or more sites of a given muscle, for example, skeletal muscle (e.g., deltoid, vastuslateralis, ventrogluteal muscle of dorsogluteal muscle, or anterolateral thigh for infants) in a subject using a needle.
In certain embodiments, the composition comprising hairpin-ended DNA molecule can be introduced to other subtypes of muscle cells. Non-limiting examples of muscle cell subtypes include skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragm muscle cells.

In certain embodiments, the compositions is delivered to multiple sites in one or more muscles of the subject. For example, the composition may be delivered by injections in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 injections sites. Such sites can be spread over the area of a single muscle or can be distributed among multiple muscles.
1004381 In some embodiments, delivery of an expressed transgene from the hairpin-ended DNA molecule, to a target tissue can also be achieved by delivering a synthetic depot comprising the hairpin-ended DNA molecule, where a depot comprising the hairpin-ended DNA molecule is implanted into skeletal, smooth, cardiac and/or diaphragm muscle tissue or the muscle tissue can be contacted matrix comprising the hairpin-ended DNA
molecule, as described herein. Such implantable matrices or substrates are described in U.S. Pat. No.
7,201,898, incorporated by reference in its entirety herein.
1004391 Methods for intramuscular injection are known to those of skill in the art and as such are not described in detail herein. However, when performing an intramuscular injection, an appropriate needle size should be determined based on the age and size of the patient, the viscosity of the composition, as well as the site of injection.
1004401 In certain embodiments, a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein is administered in the absence of a carrier to facilitate entry of hairpin-ended DNA molecule into the cells, or in a physiologically inert pharmaceutically acceptable carrier (i.e., any carrier that does not improve or enhance uptake of the capsid free, non- viral vectors into the myotubes). In such embodiments, the uptake of the hairpin-ended DNA molecule for expression of GDE protein can be facilitated by electroporation of the cell or tissue. With electroporation, electrical fields are used to create pores in cells without causing permanent damage to the cells. These pores are large enough to allow hairpin-ended DNA molecule for expression of GDE to gain access to the interior of the cell.
Over time, the pores in the cell membrane close and the cell once again becomes impermeable.
1004411 There are a number of methods for in vivo electroporation; electrodes can be provided in various configurations such as, for example, a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, needle-shaped electrodes may be inserted into the tissue, to access more deeply located cells. In either case, after the composition comprising e.g., hairpin-ended DNA molecule for expression of GDE
are injected into the treatment region, the electrodes apply an electrical field to the region. In some electroporation applications, this electric field comprises a single square wave pulse on the order of 100 to 500 V/cm. of about 10 to 60 ms duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820, made by the BTX
Division of Genetronics, Inc.

1004421 In another embodiment, a hairpin-ended DNA molecule for expression of GDE
protein is administered to the liver. The hairpin-ended DNA may also be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and portaamygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus.. The hairpin-ended DNA vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). The hairpin-ended DNA
for expression of GDE protein may further be administered intravascularly to the CNS in situations in which the blood-brain barrier has been perturbed (e.g., brain tumor or cerebral infarct).
1004431 In some embodiments, the hairpin-ended DNA for expression of GDE
protein can be administered in a liquid formulation by direct injection (e g , stereotactic injection) to the desired region or compartment in the CNS. In other embodiments, the hairpin-ended DNA
molecule can be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation.
5.9.4 Dosing 1004441 Provided herein are methods of treatment comprising administering to the subject an effective amount of a composition comprising a hairpin ended vector encoding an GDE
protein as described herein. As will be appreciated by a skilled practitioner, the term "effective amount" refers to the amount of the hairpin-ended DNA molecule composition administered that results in expression of the GDE protein in a "therapeutically effective amount" for the treatment of a disease or a disorder associated to reduced presence or function of GDE in a subject (e.g. GSDIII) .
1004451 In vivo and/or in vitro assays can optionally be employed to help identify optimal dosage ranges for use. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the person of ordinary skill in the art and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems, (e.g. patient derived fibroblasts , murine or canine models) 1004461 Hairpin ended vectors for expression of GDE protein as disclosed herein, can be administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene expression without undue adverse effects. It is desirable that the lowest effective concentration hairpin ended vector encoding GDE be utilized in order to reduce the risk of undesirable effects, such as toxicity. In some embodiments other dosages in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, and the degree to which the disorder, has developed. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, those described above in the "Administration"
section, such as direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration can be combined, if desired.
1004471 In certain embodiments, the amount (i.e. dose) of a hairpin ended vectors for expression of GDE protein as disclosed herein required to achieve a particular "therapeutic effect," will vary based on several factors including, but not limited to: the route of nucleic acid administration, the pharmaceutical carrier, the level of gene expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting expressed protein(s). One of skill in the art can readily determine a hairpin ended vector dose range to treat a patient having a disease or a disorder associated to reduced presence or function of GDE in a subject (e.g.
GSDiii) based on the aforementioned factors, as well as other factors that are well known in the art.
1004481 In general, the dosage regime can be adjusted to provide the optimum therapeutic response. For example, the hairpin ended vectors for expression of GDE protein can be repeatedly administered, e.g., several doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject vectors described herein as well as whether the said vectors are to be administered to cells or to subjects.
1004491 A "therapeutically effective dose" will fall in a relatively broad range that can be determined through clinical trials and will depend on the particular application (for example, direct ocular injections require very small amounts, while systemic injection would require large amounts). For example, for direct in vivo injection into skeletal or cardiac muscle of a human subject, a therapeutically effective dose will be on the order of from about 1 p.g to 100 g of the hairpin-ended DNA molecule. If exosomes or hybridosomes are used to deliver the hairpin-ended DNA molecule vector, then a therapeutically effective dose can be determined experimentally, but is expected to deliver from 1 ptg to about 100 g of vector. Moreover, a therapeutically effective dose is an amount hairpin-ended DNA molecule that expresses a sufficient amount of the transgene to have an effect on the subject that results in a reduction in one or more symptoms of the disease, but does not result in significant off-target or significant adverse side effects. In one embodiment, a "therapeutically effective amount" is an amount of an expressed GDE protein that is sufficient to produce a statistically significant, measurable change in expression of GSDIII biomarker or reduction of a given disease symptom. Such effective amounts can be gauged in clinical trials as well as animal studies for a given hairpin-ended DNA molecule composition. In some embodiments, a transgene encodes a catalytically active fragment of GDE. A "catalytically active fragment of GDE" is any truncated form of GDE which retains its catalytic functions.
[00450] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
[00451] For in vitro transfection, an effective amount of a hairpin-ended DNA
molecule vectors for expression of GDE protein as disclosed herein to be delivered to cells (1x106 cells) will be on the order of 0.1 to 100 pig hairpin-ended DNA molecule vector, preferably 1 to 20 pig, and more preferably 1 to 15 pig or 8 to 10 pig. Larger hairpin-ended DNA molecule vectors will require higher doses. If Hybridosomes, exosomes or lipid nanoparticles are used, an effective in vitro dose can be determined experimentally but would be intended to deliver generally the same amount of the hairpin-ended DNA molecule vector.
[00452] For the treatment of GSDIII, the appropriate dosage of a hairpin-ended DNA
molecule vector that expresses an GDE protein as disclosed herein will depend on the specific type of disease to be treated, the type of a GDE protein, the severity and course of the GSDITI disease, previous therapy, the patient's clinical history and response to the vector, and the discretion of the attending physician. The hairpin-ended DNA molecule vector encoding a GDE protein is suitably administered to the patient at one time or over a series of treatments.
Various dosing schedules including, but not limited to, single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
[00453] Depending on the type and severity of the disease or disorder, a hairpin-ended DNA molecule vector is administered in an amount that the encoded GDE protein is expressed at about 0.3 mg/kg to 100 mg/kg (e.g. 15 mg/kg- 100 mg/kg, or any dosage within that range), by one or more separate administrations, or by continuous infusion. One typical daily dosage of the hairpin-ended DNA molecule is sufficient to result in the expression of the encoded GDE protein at a range from about 15 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. One exemplary dose of the hairpin-ended DNA
molecule is an amount sufficient to result in the expression of the encoded GDE protein as disclosed herein in a range from about 10 mg/kg to about 50 mg/kg. Thus, one or more doses of a hairpin-ended DNA molecule in an amount sufficient to result in the expression of the encoded GDE protein at about 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 3 mg/kg, 4.0 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg (or any combination thereof) may be administered to the patient.
1004541 In some embodiments, a therapeutically effective dose of a hairpin-ended DNA
encoding GDE in vivo can be a dose of about 0.001 to about 500 mg/kg body weight. For instance, the therapeutically effective dose may be about 0 001-0 01 mg/kg body weight, or 0.01-0.1 mg/kg, or 0.1-1 mg/kg, or 1-10 mg/kg, or 10-100 mg/kg. In some embodiments, a hairpin-ended DNA molecule encoding GDE is provided at a dose ranging from about 0.1 to about 10 mg/kg body weight, e.g., from about 0.5 to about 5 mg/kg, from about 1 to about 4.5 mg/kg, or from about 2 to about 4 mg/kg.
1004551 In another embodiment the therapeutically effective dose of an hairpin-ended DNA encoding GDE in vivo can be a dose of at least about 0.001 mg/kg body weight, or at least about 0.01 mg/kg, or at least about 0.1 mg/kg, or at least about 1 mg/kg, or at least about 2 mg/kg, or at least about 3 mg/kg, or at least about 4 mg/kg, or at least about 5 mg/kg, at least about 10 mg/kg, at least about 20 mg/kg, at least about 50 mg/kg, or more. In some embodiments, a hairpin-ended DNA encoding GDE is provided at a dose of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 5 mg/kg, or about 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 mg/kg.
1004561 In some embodiments, the hairpin-ended DNA molecule is an amount sufficient to result in the expression of the encoded GDE protein for a total dose in the range of 50 mg to 2500 mg. An exemplary dose of a hairpin-ended DNA molecule is an amount sufficient to result in the total expression of the encoded GDE protein at about 50 mg, about 100 mg, 200 mg, 300 mg, 400 mg, about 500 mg, about 600 mg, about 700 mg, about 720 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, or about 2500 mg (or any combination thereof). As the expression of the GDE protein from hairpin-ended DNA molecule can be carefully controlled by regulatory switches herein, or alternatively multiple dose of the hairpin-ended DNA molecule administered to the subject, the expression of the GDE protein from the hairpin-ended DNA molecule can be controlled in such a way that the doses of the expressed GDE protein may be administered intermittently, e.g. every week, every two weeks, every three weeks, every four weeks, every month, every two months, every three months, or every six months from the hairpin-ended DNA molecule. The progress of this therapy can be monitored by conventional techniques and assays.
1004571 In certain embodiments, a hairpin-ended DNA molecule is administered an amount sufficient to result in the expression of the encoded GDE protein at a dose of 15 mg/kg, 30 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg or a flat dose, e.g., 300 mg, 500 mg, 700 mg, 800 mg, or higher.
1004581 In some embodiments, the expression of the GDE protein from the hairpin-ended DNA molecule is controlled such that the GDE protein is expressed every day, every other day, every week, every 2 weeks or every 4 weeks for a period of time. In some embodiments, the expression of the GDE protein from the hairpin-ended DNA molecule is controlled such that the GDE protein is expressed every 2 weeks or every 4 weeks for a period of time. In certain embodiments, the period of time is 6 months, one year, eighteen months, two years, five years, ten years, 15 years, 20 years, or the lifetime of the patient.
1004591 Treatment can involve administration of a single dose or multiple doses. In some embodiments, more than one dose can be administered to a subject. Without wishing to be bound by any particular theory or mechanism, comparison to viral vectors, multiple doses can be administered as needed, because the hairpin-ended DNA molecule does not elicit an anti-viral host immune response due to the absence of proteins of viral origin. As such, one of skill in the art can readily determine an appropriate number of doses. The number of doses administered can, for example, be on the order of 1-100, or on the order of 2-50 doses.
1004601 In certain embodiments, the interval between a first administration said hairpin-ended DNA via and second administration said may be about 0.5 hour, 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more.
1004611 Without wishing to be bound by any particular theory, the lack of typical anti-viral immune response (i.e., the absence anti-viral protein responses) elicited by administration of a composition comprising a hairpin-ended DNA molecule described herein allows the hairpin-ended DNA molecule for expression of GDE protein to be administered to a host on multiple occasions. In some embodiments, the number of occasions in which a hairpin-ended DNA molecule for the expression of GDE is delivered to a subject is in a range of 2 to 10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, a hairpin-ended DNA
molecule is delivered to a subject more than 10 times.
1004621 In some embodiments, a dose of a hairpin-ended DNA molecule for expression of GDE protein as disclosed herein is administered to a subject no more than once per calendar day (e g , a 24-hour period) In some embodiments, a dose of a hairpin-ended DNA molecule is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein is administered to a subject no more than once per calendar week (e.g., 1 calendar days). In some embodiments, a dose of a hairpin-ended DNA molecule is administered to a subject no more than bi-weekly (e.g., once in a two calendar week period).
In some embodiments, a dose of a hairpin-ended DNA molecule is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of a hairpin-ended DNA molecule is administered to a subject no more than once per six calendar months. In some embodiments, a dose of a hairpin-ended DNA
molecule is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).
1004631 In particular embodiments, more than one administration (e.g., two, three, four or more administrations) of a hairpin-ended DNA molecule for expression of GDE
protein as disclosed herein, may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.
1004641 In some embodiments, a therapeutic a GDE protein encoded by a hairpin-ended DNA molecule as disclosed herein can be regulated by a regulatory switch, inducible or repressible promotor so that it is expressed in a subject for at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 12 months/one year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years or more. In one embodiment, the expression can be achieved by repeated administration of the hairpin-ended DNA molecules described herein at predetermined or desired intervals.
1004651 The duration of treatment depends upon the subject's clinical progress and responsiveness to therapy. In one embodiment, repeated, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.
1004661 In some embodiments, the pharmaceutical compositions comprising a hairpin-ended DNA molecule for expression of GDE protein as disclosed herein can conveniently be presented in unit dosage form. A unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for droplets to be administered directly to the eye In some embodiments, the unit dosage form is adapted for administration by inhalation In some embodiments, the unit dosage form is adapted for administration by a vaporizer. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for subretinal injection, suprachoroidal injection or intravitreal injection.
1004671 In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
5.9.5 Outcome Assessments 1004681 A therapeutically effective dose can be administered in one or more separate administrations, and by different routes. As will be appreciated in the art, a therapeutically effective dose or a therapeutically effective amount is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present disclosure. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g. , treating, modulating, curing, preventing and/or ameliorating GSDIII). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., a hairpin-ended DNA molecule encoding GDE) administered to a subject in need thereof will depend upon the characteristics of the subject.
Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.
1004691 In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule as desribed herein can lead to increased liver GDE protein levels in a treated subject. In some embodiments, administering a composition comprising a hairpin-ended DNA molecule described herein results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% increase in liver GDE
protein levels relative to a baseline GDE protein level in the subject prior to treatment. In certain embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule as described herein will result an increase in liver GDE levels relative to baseline liver GDE levels in the subject prior to treatment. In some embodiments, the increase in liver GDE levels relative to baseline liver GDE levels will be at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or more.
1004701 In some embodiments, administering a composition comprising a hairpin-ended DNA molecule described herein results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in liver GDE protein levels relative to a baseline GDE
protein level in the subject prior to treatment. In certain embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule as described herein will result an increase in liver GDE levels relative to baseline liver GDE levels in the subject prior to treatment. In some embodiments, the increase in liver GDE levels relative to baseline liver GDE levels will be at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or more.
1004711 In some embodiments, a therapeutically effective dose, when administered regularly, results in increased expression of GDE in the liver as compared to baseline levels prior to treatment. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule desribed herein results in the expression of a GDE protein level at or above about 10 ng/mg, about 20 ng/mg, about 50 ng/mg, about 100 ng/mg, about 150 ng/mg, about 200 ng/mg, about 250 ng/mg, about 300 ng/mg, about 350 ng/mg, about 400 ng/mg, about 450 ng/mg, about 500 ng/mg, about 600 ng/mg, about 700 ng/mg, about 800 ng/mg, about 900 ng/mg, about 1000 ng/mg, about 1200 ng/mg or about 1500 ng/mg of the total protein in the liver of a treated subject.
1004721 In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule encoding GDE described herein will result in reduced levels of one or more of markers selected from alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), glycogen, and limit dextrin.
1004731 In some embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of ALT, AST, ALP, and/or CPK levels in a biological sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule described herein results in a reduction of ALT, AST, ALP, and/or CPK levels in a biological sample (e.g. , a plasma or serum sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% as compared to baseline ALT, AST, ALP, and/or CPK
levels before treatment. In some embodiments, the biological sample is selected from plasma, serum, whole blood, urine, or cerebrospinal fluid.
In certain exemplary embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of ALT levels, e.g., as measured in units of ALT activity/liter (U/1), in a serum or plasma sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of ALT levels in a biological sample (e.g. , a plasma or serum sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% as compared to baseline ALT levels before treatment. In an exemplary embodiment, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of ALT levels in a biological sample (e.g. , a plasma or serum sample) by at least about 50% as compared to baseline ALT levels before treatment. In a further exemplary embodiment, ALT levels are measured after fasting, e.g. , after 6, 8, 10, 12, 18, or 24 hours of fasting.

1004741 In other exemplary embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of AST levels, e.g., as measured in units of AST
activity/liter (U/1), in a serum or plasma sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA
molecule of this disclosure results in a reduction of AST levels in a biological sample (e.g. , a plasma or serum sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% as compared to baseline AST levels before treatment. In an exemplary embodiment, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of AST
levels in a biological sample (e g , a plasma or serum sample) by at least about 50% as compared to baseline AST levels before treatment. In a further exemplary embodiment, AST
levels are measured after fasting, e.g. , after 6, 8, 10, 12, 18, or 24 hours of fasting.
1004751 Measurements of ALT, AST, ALP, and/or CPK levels can be made using any method known in the art, e.g., using a Fuji Dri-Chem Clinical Chemistry Analyzer FDC 3500 as described in Liu et al. , 2014, Mol Genet and Metabolism 111: 467-76.
1004761 In other exemplary embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of glycogen levels in a biological sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of glycogen accumulation in a biological sample (e.g. , a liver sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%
as compared to baseline glycogen levels before treatment. In some embodiments, the biological sample is a portion of an organ selected from liver, heart, diaphragm, quadriceps, and gastrocnemius. In an exemplary embodiment, the biological sample is a liver section, e.g., a section of hepatocytes.
1004771 In other exemplary embodiments, a therapeutically effective dose, when administered regularly, results in a reduction of limit dextrin levels in a biological sample. In some embodiments, administering a therapeutically effective dose of a composition comprising a hairpin-ended DNA molecule of this disclosure results in a reduction of limit dextrin accumulation in a biological sample (e.g. , a liver sample) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%
as compared to baseline limit dextrin levels before treatment. In some embodiments, the biological sample is a portion of an organ selected from liver, heart, diaphragm, quadriceps, and gastrocnemius. In an exemplary embodiment, the biological sample is a liver section, e.g., a section of hepatocytes. In a further exemplary embodiment, a therapeutically effective dose, when administered regularly, results in at least a 50%, 60%, 70%, or 80% reduction of limit dextrin levels in a liver sample as compared to baseline limit dextrin levels before treatment.
1004781 In further embodiments, a therapeutically effective dose, when administered regularly, delays the onset of liver fibrosis in a treated subject. In some embodiments, a therapeutically effective dose, when administered regularly, slows the development of liver fibrosis or reduces the amount of liver fibrosis in a subject afflicted with GSDIII.
5.10 Kits 1004791 In another aspect, provided herein are kits for expressing human GDE
in vivo, e.g., in a human patient. In some embodiments, a kit provided herein comprises 0.1-500 mg of one or more DNA molecules provided herein. In some embodiments, the kit further comprises a device for administering the dose. In some embodiments, the device is an injection needle.
1004801 All patent applications, publications (patents and patent applications, scientific literature, or any other publications), patents, GenBank citations and other database citations, webpage disclosures, commercial catalogs, and other references cited herein are incorporated by reference in their entirety.
6. Examples 1004811 A number of embodiments have been described. Nevertheless, it will be understood that various examples in this Section (i.e., Section 6) describes specific embodiments herein solely for the purpose of illustration and do not limit the scope as described in the claims or the disclosure. Various modifications can be made without departing from the spirit and scope of what is provided herein.
6.1 Example 1 ¨ Production of Plasmids Encoding the Vector 1004821 The nucleic acid sequences encoding the AGL expression cassette were designed in silico. Construct 1 encodes for a modified left ITR, a human PGK promoter, a AGL ORF , bGH poly (a), a right ITR and a double restriction sites for nicking endonuclease 113 base pairs downstream of the right ITR
(TGCGCGACTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCG
GGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGTCGCGCAGAGAGGTTAAAAC
CAACTAGACAACTTTGTATATCTAGAGTTGGGGTTGCGCCTTTTCCAAGGCAGCC
CTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCG
GCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGAT
CTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGT
CGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCA
CGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGC
C GAC C GC GATGGGC T GTGGC CAATAGC GGC T GC TC AGC AGGGC GC GC C GAGAGC
AGC GGCC GGGAAGGGGC GGT GC GGGAGGC GGGGT GT GGGGC GGTAGT GTGGGC
CCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCG
GCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGCAAGTTTGT
AC AAAAAAGC GC GCCGCCAT GGGC CAT AGC AAAC AAATAC GC ATAC TGC TGC TC
AATGAGATGGAGAAACTTGAGAAAACACTGTTTCGCCTGGAGCAGGGATACGAA
CTTCAATTTAGATTGGGACCTACCCTICAAGGGAAGGCCGTGACTGTTTACACTA
ACTATCCTTTCCCCGGTGAGACCTTCAACCGGGAGAAGTTTCGGAGCTTGGACTG
GGAGAACCCCACTGAGCGAGAGGACGACAGTGACAAGTATTGCAAGCTGAACCT
TCAGCAGTCCGGGAGTTTCCAATACTACTTTCTCCAGGGTAACGAAAAGTCTGGC
GGTGGCTATATTGTCGTCGATCCTATACTGAGGGTCGGGGCAGACAACCACGTTC
TGCCGCTCGATTGCGTCACGCTGCAAACGTTCTTGGCAAAATGCCTTGGGCCCTT
CGACGAGTGGGAGAGCCGGCTCCGTGTCGCTAAAGAGAGTGGTTATAATATGAT
CCACTTCACTCCTCTGCAAACCCTGGGGCTCAGCAGATCCTGTTATAGCCTGGCA
AACCAACTTGAGCTGAACCCCGATTTCTCCAGGCCCAACCGTAAATACACTTGGA
ACGACGTGGGGCAACTTGTCGAGAAGCTGAAGAAAGAGTGGAACGTCATCTGCA
T C AC C GAC GT GGTGTATAAC C AC AC AGC C GC C AAC TC C AAGT GGAT TC AAGAGC
ACCCCGAGTGCGCGTACAACCTGGTCAACTCACCGCATCTTAAGCCGGCTTGGGT
GCTGGATCGGGCTCTGTGGAGATTTTCTTGCGACGTGGCTGAGGGTAAGTACAAG

GAGAAAGGGATCCCAGCGCTGATCGAGAACGACCATCACATGAACTCTATTCGC
AAGATTATATGGGAAGACATCTTCCCGAAACTGAAGCTGTGGGAGTTCTTTCAGG
TGGACGTGAATAAGGCCGTAGAACAGTTCAGGCGGTTGCTGACCCAGGAGAACA
GAAGGGTGACGAAAAGCGACCCCAATCAGCATCTCACTATAATCCAGGACCCCG
AGTATCGGCGATTCGGGTGCACCGTTGACATGAATATAGCTCTCACAACATTTAT
TCCCCACGATAAAGGACCGGCCGCTATAGAGGAGTGTTGCAACTGGTTCCACAA
GCGGATGGAAGAGCTGAACTCCGAAAAGCACCGCCTTATCAATTACCACCAAGA
GCAAGCCGTGAACTGTCTGCTCGGGAACGTCTTCTACGAGAGGCTCGCCGGGCA
CGGCCCGAAGCTGGGCCCAGTTACCCGCAAACACCCACTGGTGACTAGGTACTT
CACCTTTCCCTTCGAGGAAATCGATTTTAGCATGGAAGAGAGTATGATCCATCTC
CCCAACAAGGCGTGCTTCCTCATGGCCCATAACGGCTGGGTGATGGGCGACGAC
CCGTTGCGTAATTTCGCGGAGCCAGGAAGCGAGGTCTATCTGCGGCGCGAGCTC
ATCTGTTGGGGAGATTCCGTGAAACTTCGATACGGAAACAAGCCCGAAGATTGC
CCCTACCTGTGGGCTCATATGAAGAAGTATACCGAGATTACCGCTACATACTTTC
AAGGCGTTAGGTTGGACAATTGTCATTCTACCCCGTTGCATGTGGCCGAATATAT
GCTCGACGCCGCCAGAAACCTGCAACCAAACCTGTACGTGGTGGCAGAGCTCTT
TACTGGGTCAGAGGACTTGGATAACGTGTTCGTCACACGACTTGGGATATCAAGT
CTTATTCGGGAAGCTATGTCTGCCTACAACTCCCACGAGGAAGGACGCCTGGTGT
ATCGTTACGGTGGGGAGCCCGTGGGGAGTTTCGTGCAACCATGCCTCAGGCCTCT
GATGCCTGCCATCGCGCACGCACTTTTCATGGACATCACTCACGACAACGAATGC
CCCATAGTTCACAGGAGTGCCTACGACGCCCTGCCTTCAACAACCATCGTCAGCA
TGGCCTGCTGCGCCAGTGGCAGCACTCGCGGGTACGACGAGCTGGTCCCACACC
AAATCAGCGTTGTCTCCGAGGAGAGATTCTATACCAAATGGAACCCGGAAGCCC
TGCCCTCTAATACTGGAGAGGTGAACTTTCAGAGTGGGATCATCGCTGCACGGTG
CGCAATTTCCAAGTTGCACCAAGAACTCGGCGCAAAAGGATTCATCCAAGTATA
CGTCGACCAGGTGGACGAGGATATCGTTGCCGTTACCCGTCATTCCCCAAGTATT
CACCAATCCGTCGTAGCAGTTTCACGCACCGCATTTCGGAACCCAAAGACCAGTT
TCTATTCCAAAGAGGTTCCGCAGATGTGTATTCCCGGGAAGATCGAGGAAGTCGT
ACTCGAAGCACGAACAATCGAACGAAATACTAAGCCATACCGTAAAGACGAAA
ACTCCATTAACGGCACCCCTGACATAACCGTGGAGATCCGCGAGCACATACAAC
TCAACGAGAGCAAGATCGTGAAGCAGGCAGGGGTGGCGACTAAGGGACCTAAC
GAGTACATCCAGGAGATCGAGTTCGAGAATCTGAGCCCCGGTTCAGTCATAATTT
TCCGAGTGTCCTTGGACCCCCACGCCCAGGTGGCAGTGGGCATCCTGCGGAACC
ACTTGACGCAGTTTTCTCCCCATTTCAAGAGTGGGTCCCTGGCCGTGGATAACGC

TGACCCCATCCTTAAGATCCCCTTC GCCAGTTTGGCAAGTCGCCTGACCCTTGCG
GAAC TCAAC CAAAT T TTGTATAGATGC GAGAGTGAGGAGAAAGAGGAC GGC GGC
GGATGTTACGATATCCCTAATTGGAGTGCACTGAAGTACGCCGGGTTGCAGGGG
CTTATGAGTGTCCTTGCTGAGATCCGTCCCAAGAACGATCTTGGTCACCCCTTCT
GCAACAACC TGAGGAGCGGT GACTGGAT GAT CGAT TACGTATC TAATAGACTGA
TAAGTAGGTCC GGCAC GATAGCCGAGGTGGGCAAGT GGC TGCAAGCCATGT TC T
TTTATTTGAAACAAATTCCCAGATATTTGATTCCTTGCTATTTCGACGCCATCCTG
ATCGGAGCGTACACGACACTGTTGGACACTGCCTGGAAACAAATGTCCAGTTTC
GTGCAAAAC GGGTCTACAT TCGT TAAGCATT TGAGCCTGGGGAGC GTACAGCTCT
GCGGCGTCGGGAAGTTTCCCTCACTTCCTATACTGTCTCCAGCACTGATGGACGT
GCCCTACCGTCTGAACGAAATTACCAAGGAGAAAGA ACAGTGCTGCGTCAGCCT
CGCAGCCGGGCTCCCCCACTTCTCTTC CGGAATATTTCGGTGTTGGGGACGCGAC
ACATTCATCGCTCTCCGCGGCATCCTCTTGATCACGGGGAGATA CGTGGAAGCTC
GGAACATAATATTGGCCTTCGCCGGAACGCTTAGACACGGCCTTATACCCAACCT
GT TGGGC GAGGGCATCTACGC TCGT TATAACTGCC GCGAC GCCGTC TGGTGGTGG
CTTCAATGCATTCAAGACTATTGCAAGATGGTGCC CAACGGGCTGGATATCCT GA
AATGTCC TGTGTCACGGATGTACC CCAC CGACGACAGCGC CC CACTCCCGGC CGG
GACGCTCGACCAACCTCTGTTCGAGGTGATCCAAGAGGCCATGCAGAAGCATAT
GCAAGGAATCCAATTTCGTGAGCGCAACGCCGGACCACAAATCGACCGCAATAT
GAAAGATGAGGGGTTCAACATCACAGCC GGT GTC GAC GAGGAGAC GGGC TTC GT
GTACGGTGGCAACAGGTTTAACTGCGGGACTTGGATGGACAAGATGGGCGAGAG
TGATC GAGCGAGGAATCGAGGCAT TC CC GCTACC CCAC GCGAC GGCAGC GCTGT
CGAGATCGTTGGGCTCTCAAAGTCCGCGGTCAGGTGGCTGTTGGAGCTGTCTAAG
AAGAACATCTTTCCCTACCACGAGGTAACGGTCAAGAGGCACGGTAAAGCCATC
AAAGTGAGCTACGACGAATGGAATCGTAAGATTCAGGATAATTTCGAGAAACTC
TTCCACGTATCTGAGGATCCATCCGACCTCAACGAGAAACACCCCAACTTGGTGC
ATAAGAGAGGGATTTATAAGGACAGTTACGGCGCCTCTAGCCCCTGGTGCGATT
ACCAACTGAGACCCAACTTCACAATCGCCATGGTCGTCGCTCCAGAATTGTTCAC
CACTGA GA AGGCCTGGA A GGCACTGGA A ATCGCGGA GA AGA AGCTGTTGGGGC
CAC TC GGTATGAAGAC GCTGGACCC GGACGACATGGTGTAT TGC GGTATCTACG
ATAACGCCTTGGATAACGATAATTATAACCTCGCAAAGGGCTTTAACTACCATCA
GGGCCCCGAATGGCTTTGGCCGATAGGTTACTTCTTGCGCGCCAAACTTTACTTC
TCTAGGCTGATGGGACCCGAAACAACCGCCAAAACAATCGTACTCGTGAAGAAC
GTGTTGAGTAGGCAC TAC GTGCAC CTC GAAAGGAGC CCATGGAAGGGGCTGCC T

GAGCTCACAAACGAAAACGCACAATATTGCCCCTTTTCATGCGAGACCCAGGCA
TGGAGCATCGCCACCATACTGGAAACCCTGTACGACTTGTGATCCTAGAGCTCGC
ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTT
GACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCA
TCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACA
GCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGGGCGCTAGC
GCAGGAACCCCTTTTAATGGAGTTGGCGAGTCCCTCTCTGCGCGCTCGCTCGCTC
ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCC
TCAGTGAGCGAGCGAGCGCGCAGAGATCGACTCCTCGGCCACTTGGAGGGGCCG
GGGGGACGACGCAATCTGGAGTGGAAAGAACCCCCGTCTATGCGGCTTAAAGCA
CGGCCAGGGAATAGTGGATCAAGTGTACTGACATGTGCCGGAGTCCCTCCATGC
CCAGATCGACTCCCTCGAGATATATGGATCC (SEQ ID NO:180).
1004831 Construct 1 was synthetized and cloned into a pUC57 backbone (plasmid 1) by a commercial DNA synthesis vendor.
1004841 Construct 2 was synthesized and circularized with a synthetic backbone containing several double nicking sites between the insert, the antibiotic resistance and the origin to produce plasmid 2.
1004851 Backbone 1:
AAGCTTAGCTTCAATAGCTGCAATGCATTGCGGAGTCACATTCGCGACTCCGCGG
AACC CC TATTTGTTTATTTTTC TAAATACATTCAAATATGTATC C GC TCATGAGAC
AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC
AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG
CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCGC
GCGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG
CC CCGAAGAACGTTTTC CAATGATGAGCACTTTTAAAGTTC TGCTGTGTGGCGCG
GTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATTCACTATT
CTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATG
GCATGACAGTACGCGAATTATGCAGTGCTGCCATTACCATGAGTGATAACACTGC
GGCC A ACTT ACTTCTGAC A ACGATCGGAGGACCGA A GGA GCTGACCGCTTTTTTG
CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAAT
GAAGC CAT CC CAAAC GACGAGC GTGAC ACC ACGAT GCC T GTAGC AATGGC AACA
ACGTTGCGCAAATTATTAAC TGGCGAAC TGC TTAC TC TAGC TTC CCGGC AAC AAT
TAATCGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG

CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC
TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAG
ATAGGTGCCTCACTGATTAAGCATTGGTAAAGTCAAAAGCCTCCGGTCGGAGGC
TTTTGACTGCAATGCATTGCCTGTCAACTCATCATTTTTAACAGCTGATGACCAA
AATCCCGCAATGCATTGCGTTCCTCGATCTTCTTGAGATCCTTTTTTTCTGCGCGT
AATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG
GATCAAGAGCTACCAACTCTITTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG
ATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT
CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC
CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT
AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG
CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC
ACGCTTCCCGAAGGGAGAA AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG
AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG
TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAG
GGGGGCGGAGCCTATGGAAAACGCCAGCGAGTCACAGCTGCGACTCCCTGGCCT
TTTGCAATGCATTGCGGCCTTTTGGGAATTC (SEQ ID NO: 182) 1004861 Plasmids 1 & 2 were transformed and then amplified overnight in the NEBstable or MDS-42 strain followed by plasmid isolation using commercial plasmid isolation kit (Nucleobond Xtra Maxi Plus EF (Macherey Nagel)) and dissolved in TE buffer.
1004871 For construct 1: To induce nicks on construct 1, the nicking endonuclease Nt.BstNBI (6.2U/ g DNA) was added to the isolated construct 1 in lx Neb3.1 Buffer and incubated at 55 C for one hour. The reaction mix containing the nicked plasmid was then heated to 95 C on a thermo shaker for 10 min, in order to dissociate the 1TR
flanked transgene from the plasmid back bone and the mix was then left to cool to room temperature for 30 min to allow for ITR folding at the single stranded overhangs ends. The reaction mix was then supplemented with both the restriction enzyme PvuII and RecBCD
Exonuclease V
(0.157U and 0.625U per [tg of nicked plasmid, respectively) as well as adenosine triphosphate (final concentration of 1mM). The reaction mix was then placed on a shaker at 37 C for 120 min to allow for the restriction enzyme to cleave the backbone fragment and the exonuclease to digest backbone fragments. The exonuclease generally does not digest linear fragments protected by closed ends. Finally, the reaction mix was purified using Takara NucleoSpin Gel and PCR clean-up kit and remaining ITR flanked vector was eluted according to the manufacturer's instructions.

1004881 For construct 2: To induce nicks and linearize construct 2, the nicking endonuclease nb.BsrDI (0.5U/pg DNA) was added to the isolated construct 2 in lx Neb3.1 Buffer and incubated at 55 C for 120 min. The reaction mix containing the nicked construct 2 was then heated to 95 C on a thermocycler for 3 min in order to dissociate the ITR flanked transgene from the plasmid back bone and subsequently cooled down to 40 C in the thermocycler with a slope of 0.05 C/s. The reaction mix was then supplemented with Exonuclease V (2.5 U/[ts of DNA) as well as adenosine triphosphate (final concentration of 1mM). The reaction mix was then placed on a shaker at 37 C for 120 min to allow for the restriction enzyme to cleave the backbone fragment and the exonuclease to digest backbone fragments. The exonuclease generally does not digest linear fragments protected by closed ends. Finally, the reaction mix was purified using a Takara NucleoSpin Gel and PCR clean-up kit and remaining ITR flanked vector was eluted according to the manufacturer's instructions 1004891 Nicked, de/renatured and digestion resistant DNA products were visualized by native agarose gel electrophoresis.
1004901 For construct 1, the agarose gel (FIG. 6C) shows the nicked plasmid in lane 3, the de/renatured DNA products in lane 4 and the single band of digestion resistant vector in lane 8.
6.2 Example 2 Transfection of LNPs and Hybridosomes 1004911 Lipid nanoparticles were prepared on a NanoassemblrTM microfluidic system (Precision NanoSystems) according to the manufacturer's instructions.
Depending on the desired formulation, an ethanol solution similar to that of the preformed vesicle approach, consisting of an ionazible lipid (e.g. MC3 ), a zwitterionic lipid (e.g., distearoylphosphatidylcholine (DSPC), dioleoylglycerophosphocholine (DOPC), a component to provide membrane integrity (such as a sterol, e.g., cholesterol) and a conjugated lipid molecule (such as a PEG-lipid, e.g., 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000 ("PEG-DMG")) at the appropriate molar ratio (e.g. 40:40:18:2), was prepared at concentrations of 10 mM total lipid. Furthermore, an aqueous DNA solution with a DNA to lipid w/w ratio of approximately 14 was prepared in 25 mM acetate buffer at pH 4Ø Depending on the total volume of production 1 and 3 ml syringes where used to create the inlet stream with a total flow rate of 12 ml/min. For each formulation the aqueous DNA solution was mixed with the ethanol-lipid solution with a flow rate ratio of 3:1 (Aq:Et) at room temperature. The product was then dialyzed against PBS to remove the residual ethanol as well as to raise the pH
to 7.4.

1004921 For exosome production, cells were grown in stirred bioreactors in perfusion mode and exosome isolation was performed by tangential flow filtration followed by Captocore 700 liquid chromatography as described in Nordin et al Methods in Molecular Biology, vol 1953. Humana Press, New York, NY (2019), which is herein incorporated in its entirety by reference.
1004931 Differentiated non-dividing HepRG cells were plated into 96 well plates and maintained in HepaRGTM Maintenance/Metabolism media.. The cells were grown at 37 C in a 5% CO2 -humidified incubator. Cells were transfected with 11 fmol hairpin ended DNA
vector described herein encoding for secreted turboluc. Transfection was mediated using Hybridosomes generated by fusing exosomes with lipid nanoparticles as outlined in US15/112,180. As a comparison, cells additionally were transfected with lipid nanoparticles.
A sample of supernatant was removed from transfected cells at different time points and the remaining medium was exchanged for fresh medium Levels of luciferase expression level in the supernatant was determined using the Gluc Glow Assay kit (NanoLight Technology) according to the manufacturer's instructions. This was repeated at several time points over 4 weeks and the expression levels are depicted in FIG. 10A.
6.3 Example 3: Expression in dividing and non-dividing cells 1004941 Constructs were generated to include an open reading frame encoding the Turboluc reporter gene into the expression cassette flanked by two ITRs.
Expression of secreted Turboluc from the vectors over time was determined based on luciferase activity.
1004951 In detail, dividing human embryonic kidney cells (HEK-2931) were cultured in DMEM (10 % FCS, 1 % pen/strep) and 2 mM stable Glutamine and differentiated non-dividing HepRG cells were maintained in HepaRGTM Maintenance/Metabolism media.

1004961 As described in Example 2, luciferase expression level was determined at different time points for non-dividing cells (FIG. 10B) and dividing cells (FIG 10C).
Luciferase activity was determined by measuring the luminescence using a SynergyMX plate reader (BioTek). For the analysis of background, bioluminescence from untreated cells was measured following the protocol described in Example 2 above. As seen in FIG.
10B, for non-dividing cells transfected with construct 3 encoding secreted Turboluc, luciferase activity remains stable over 4 weeks. As seen in FIG. 10C luciferase activity peaks in dividing cells on day 2 and gradually decreases over time. As a direct comparison, equimolar amounts of full circular plasmids encoding construct 3 were also transfected and as seen in FIG. 10B and FIG. 10C ,luciferase activity decreased over-time in both dividing and non-dividing cells.
6.4 Example 4: GDE Activity assays 1004971 For the GDE assay, 13-limit dextrin (Megazyme) was used as a substrate to quantify the combined enzymatic activities of glucantransferase and a-1,6-glucosidase of GDE. Fibroblast from a GDSIII patient (Coriell GM00226) a healthy subject (OUMS-36T-2F)in DMEM/F12 + 15% FBS. One million cells were detached with trypsin and washed thrice with cold PBS and pelleting at 300g. The cell pellet was lysed in 10 mM
Citrate, 100 mM NaCl, 0.1 % Tween-20, pH 6.0 and the lysate was incubated with j3-Limit dextrin (5%, Megazyme) at 30 C for 16 hours. The amount of released glucose in the supernatant of each sample was quantified using a glucose HK kit (Megazyme). Results are shown in Table 22 below.
Table 22: Remaining Glucose Activity Name mean SD Remaining activity Remaining activity according to supplier [ [ [0/01 roi GM00226 0.6 0.2 5.7 <10 OUMS-36T-2F 5.3 0.4 1004981 For testing the GDE expression, GM00226 cells or C2C12 cells (3x104/well) were seeded in a 96-well plate. After 24 hours, cells were transfected with 10Ong, 50ng or lOng of hairpin-ended DNA vector (purified construct 1 of example 1) encoding for GDE.
After 48 hours, GDE activity was measured was assayed by washing the cells with ice cold PBS, lysing the cell in 10 mM Citrate, 100 mM NaCl, 0.1 % Tween-20, pH 6.0 and then the lysate was incubated with 13-Limit dextrin (5%, Megazyme) at 30 C for 16 hours. The amount of released glucose in the supernatant of each sample was quantified using a glucose 1-IK kit (Megazyme). The amount to glucose released is depicted in FIGs. 8A
and 8B.
6.5 Example 5: Glycogen Content After Starvation 1004991 GSDIII patient derived and wildtype (OUMS) fibroblasts were grown in a 96 well in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The cells were lipofected with 10 fmol of either a hairpin-ended DNA
molecule encoding GDE or GFP as a control. After 48h, medium was removed, and cells were washed twice with PBS. Cell starvation was performed by incubation of fibroblasts for lh or 4h in glucose-free DMEM, supplemented with 2mM stable glutamine.
1005001 After glucose starvation, the supernatant was removed. Cells then were treated with HC1 0.6M and triton. Therefore, 26 uL PBS, 5 uL HC1 and 5 uL of Triton (10% stock) were added to cells and incubated under constant shaking.
1005011 The inactivation/lysis was stopped by the addition of 3.6 uL Tris (1M, pH 10.7), after 30 sec. of shaking, the glycogen degrading enzymes: a- Amylase (16.6 Units), Amyloglucosidase (0.066 Units) and a-Glucosidase (6 Units) were added to wells. The plate then was then incubated at 37.5 C for lh.
1005021 Glucose detection (Promega Glucose Glo Assay) reagent was prepared according to the manufacturer protocol. 10 uL of each sample was removed from the plate and transferred to a detection plate. 40 uL of PBS as well as 50 .1_, of the detection reagent was added. Luminescence was recorded on a plate reader. The amount of glycogen converted into glucose detected by the Glucose Glo Assay is depicted in FIGs. 9A and 9B.
Despite glucose starvation, the GSDIII patient derived fibroblasts showed a high glycogen content when treated with GFP control and a low content when treated with the GDE
construct. Wild type GDE expressing fibroblasts contained similar glycogen contents after glucose starving, after both treatment with GFP or GDE encoding DNA constructs.
6.6 Example 6 : Treatment of GSDIII with hairpin-ended GDE DNA
constructs A hairpin-ended DNA encoding GDE, described herein, is deemed useful for treatment of GSDIII when expressed as a transgene. A subject presenting with GSDIII is administered a hairpin ended DNA-based vector that encodes GDE intravenously at a dose sufficient to deliver and maintain a therapeutically effective concentration of GDE protein.
Following treatment, the subject is evaluated for improvement in symptoms of GSDIII. The ability of the hairpin ended DNA-based vector to induce normoketonemia after 12 hours of fasting is determined.
6.7 Example 7: Treatment of GSDIII in animals models with GDE
A human GDE-based vector is deemed useful for treatment of GSDIII when expressed as a transgene. An animal model for GSDIII, for example an animal model described in Liu, K.M. et al; Mol. Genet. Metab. 2014, 111, 467-476 (mice), Pagliarani, S et al.
Biochim. Et Biophys. Acta 2014, 1842, 2318-2328 (mice), Vidal, Pet al; Mol. Ther. J. Am. Soc. Gene Ther.
2018, 26, 890-901 (mice), or in Gregory, B.L et al. Glycogen storage disease type Ina in curly-coated retrievers.

J. Vet. Intern. Med. 2007, 21, 40-46 (dog), is administered a hairpin-ended DNA molecule described herein that encodes GDE intravenously at a dose sufficient to deliver and maintain a therapeutically effective concentration of GDE protein. Following treatment, the animal is evaluated for improvement in symptoms consistent with the disease in the particular animal model. The ability of the hairpin ended DNA-based vector to induce normoketonemia after 12 hours of fasting is determined.
6.8 Example 8: Clinical Protocol Treatment of GSDIII
1005031 The following example sets out a proposed protocol that may be used to treat human subjects with a hairpin-ended DNA molecule encoding GDE to treat GSDIII.
1005041 Patient Population. Patients to be treated may include males or females who have:
= Confirmed historical diagnosis of GSDIII based on pathogenic mutations in the AGL
gene on both alleles or GDE deficiency based on biopsy of liver, muscle, or fibroblasts = Documented history of >1 hypoglycemic event with blood glucose <60 mg/dL
(<3.33 mmol/L) = Patient's GSDIII disease is stable as evidenced by no hospitalization for severe hypoglycemia during the 4-week period preceding the screening visit = Key Exclusion Criteria:
o Screening or Baseline (Day 0) blood glucose level <60 mg/dL (<3.33 mmol/L) o Liver transplant, including hepatocyte cell therapy/transplant o Presence of liver adenoma >5 cm in size o Presence of liver adenoma >3 cm and <5 cm in size that has a documented annual growth rate of >0.5 cm per year o Gene Therapy 1005051 A hairpin-ended DNA molecule comprising a human GDE expression cassette encapsulated in a lipid nanoparticle is used for treatment. The LNP allows for efficient expression of the GDE protein in the liver following IV administration. The hairpin-ended DNA molecule a comprises double stranded GDE expression cassette flanked by inverted terminal repeats..
1005061 From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a human patient, the method comprising administering to the patient a biocompatible carrier (hybridosome) or lipid nanoparticle, wherein the hybridosome or the lipid nanoparticle comprises a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof.

2. A method for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a human patient, the method comprising administering to the patient a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, wherein the DNA molecule is contained within a single delivery vector.

3. A method for treating a disease associated with reduced activity of GDE
in a human patient, the method comprising the steps of (i) administering a first dose of a DNA
molecule comprising an expression cassette comprising a transgene encoding human GDF, or a catalytically active fragment thereof to the patient and (ii) administering a second dose of the DNA molecule to the patient.

4. The method of claim 3, wherein the first dose of the DNA molecule is administered to the patient at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months before the second dose of the DNA molecule.

5. The method of claim 3, wherein the first dose of the DNA molecule is administered to the patient at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, or at least 20 years before the second dose of the DNA molecule.

213 The method of any one of claims 3-5, wherein the first dose of the double-stranded DNA molecule and the second dose of the DNA molecule contain the same amount of the DNA molecule.

7 The method of any one of claims 3-5, wherein the first dose of the DNA
molecule and the second dose of the DNA molecule contain different amounts of the DNA
molecule.

8. The method of claim 3, the method further comprising administering one or more additional doses of the DNA molecule.

9. The method of claim 8, wherein the DNA molecule is administered once weekly, biweekly, or monthly.

10. The method of claim 8 or 9, wherein the DNA molecule is administered to the patient about every 6 months, about every 12 months, about every 18 months, about every 2 years, about every 3 years, about every 5 years, about every 10 years, about every 15 years or about every 20 years.

11. The method of claim 8 to 10, wherein the DNA molecule is administered to the patient for the duration of the life of the patient

12. The method of claim 1 to 11, wherein the patient is an adult patient.

13. The method of claim 1 or 11, wherein the patient is a pediatric patient.

14. The method of any one of claims 3-11, wherein the patient is a pediatric patient when the first dose of the DNA molecule is administered.

15. The method of claim 13 or 14, wherein the pediatric patient is an infant.

16. The method of claim 13 or 14, wherein the pediatric patient is about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, or about 18 years old

17. The method of any one of claims 1-16, wherein the disease is Glycogen Storage Disease (GDS) Type III (GSDIII).

18. The method of any one of claims 1-17, wherein the disease is GSDIIIa, GSDIIIb, GSDIIIc, and GSDIIId.

19. The method of any one of claims 1-18 wherein the transgene comprises a sequence that is at least 60%, at least 70%, at least 80% or at least 90%
identical to the sequence set forth in SEQ ID NO: 174, 175, 178, or 179.

20. The method of any one of claims 1-19, wherein the method results in an improvement of one or more of the following clinical symptoms of GSDIII:
fasting intolerance, exercise intolerance, growth failure, myopathy, muscle weakness, and hepatomegaly.

21. The method of any one of claims 1-19, wherein the method results in a reduction in the number of hypoglycemic episodes per year of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% in the patient

22. The method of any one of claims 1-19, wherein the method results in an improvement in liver function of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%
in a patient as determined by liver function tests.

23. The method of any one of claims 1-19, wherein the method results in a reduction in the number of hyperlipidemic episodes per year of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% in the patient.

24. The method of any one of claims 1-19, wherein the method results in a clinical improvement of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater than about 95% as measured by one or more of the following metabolic markers: glucose, lactate, ketones, creatine phosphokinase, uric acid, lipids or ketones.

25. The method of any one of claims 1-19, wherein the method results in a clinical improvement of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater than about 95% as measured by the levels of urinary glucose tetrasaccharide (G1c4) in the patient.

26. The method of any one of claims 1-19, wherein the method results in GDE

protein activity of about 1-10%, about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, or about 80-90% of the biological activity level of the native GDE protein.

27. The method of any one of claims 1-26, wherein the DNA molecule is detectable in the hepatocytes of the patient by quantitative real-time PCR.

28. The method of any one of claims 1-27, wherein the method results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 95% decrease in limit dextrin accumulation in a biological sample (e.g., a liver sample) from the patient.

29. The method of any one of claims 1-26, wherein the DNA molecule is detectable in the muscle tissue of the patient by quantitative real-time PCR.

30. The method of any one of claims 1-27, wherein the method results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 95% decrease in limit dextrin accumulation in a biological sample (e.g., a muscle sample) from the patient.

31. A double-stranded DNA molecule comprising in 5' to 3' direction of the top strand:
a.
a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
b. an expression cassette comprising a transgene encoding human GDE
or a catalytically active fragment thereof; and c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

32. A double strand DNA molecule comprising in 5' to 3' direction of the top strand:
a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
b. an expression cassette comprising a transgene encoding human GDE
or a catalytically active fragment thereof; and c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

33. A double-stranded DNA molecule comprising in 5' to 3' direction of the top strand:
a.
a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;

b. an expression cassette comprising a transgene encoding human GDE
or a catalytically active fragment thereof; and c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

34. A double strand DNA molecule comprising in 5' to 3' direction of the top strand:
a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
b. an expression cassette comprising a transgene encoding human GDE
or a catalytically active fragment thereof; and c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

35. The DNA molecule of any one of claims 31 to 34, wherein the DNA
molecule is an isolated DNA molecule.

36. The DNA molecule of any one of claims 31 to 35, wherein the first, second, third, and fourth restriction sites for nicking endonuclease are all restriction sites for the same nicking endonuclease.

37. The DNA molecule of any one of claims 31 to 35, wherein the first and the second inverted repeats are the same.

38. The DNA molecule of any one of claims 31 to 35, wherein the first and/or the second inverted repeat is an ITR of a parvovirus.

39. The DNA molecule of any one of claims 31 to 35, wherein the first and/or the second inverted repeat is a modified ITR of a parvovirus.

40. The DNA molecule of claim 38 or 39, wherein the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus.

41, The DNA molecule of claim 39 wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99%
identical to the ITR of the parvovirus.

42. The DNA molecule of any one of claims 38 to 41, wherein the ITR
comprises a viral replication-associated protein binding sequence ("RABS").

43. The DNA molecule of claim 42, wherein the RABS comprises a Rep binding sequence.

44. The DNA molecule of claim 42, wherein the RABS comprises an NS1-binding sequence

45. The DNA molecule of any one of claims 38 to 41, wherein the ITR does not comprise a RABS.

46. The DNA molecule of any one of claims 31 to 45, wherein the transgene comprises a sequence of SEQ ID NO: 174, 175, 178, or 179.

47, The DNA molecule of claim 31 or 35, wherein the a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat, b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat; and/or d. the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat.

48. The DNA molecule of claim 32 or 35, wherein the a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat; and/or d. the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat.

49. The DNA molecule of claim 33 or 35, wherein the a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat; and/or d. the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat.

50. The DNA molecule of claim 34 or 35, wherein the a. the first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the first inverted repeat;
b. the second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the first inverted repeat;
c. the third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of the second inverted repeat; and/or d. the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of the second inverted repeat.

1 The DNA molecule of any one of claims 47 to 50, wherein the nick is inside the inverted repeat.

52. The DNA molecule of any one of claims 47 to 50, wherein the nick is outside the inverted repeat.

53. The DNA molecule of any one of claims 31 to 52, wherein the DNA
molecule is a plasmid.

54. The DNA molecule of claim 53, wherein the plasmid further comprises a bacterial origin of replication.

55. The DNA molecule of claim 53, wherein the plasmid further comprises a restriction enzyme site in the region 5' to the first inverted repeat and 3' to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and the region between the first and second inverted repeats.

56. The DNA molecule of claim 55, wherein the cleavage with the restriction enzyme results in single strand overhangs that do not anneal at detectable levels under conditions that favor annealing of the first and/or second inverted repeat.

57. The DNA molecule of claim 53, wherein the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are:
a. on opposite strands; and b. create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter-or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat.

58. The DNA molecule of claim 57, wherein the fifth and the sixth nick are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart.

59. The DNA molecule of claim 57, wherein the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are all target sequences for the same nicking endonuclease.

60. The DNA molecule of any one of claim 31 to 59, wherein the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N. BspD6I; Nb.
Mva1269I; Nb.
BsrDI, Nt. BtsI, Nt. BsaI, Nt. Bpul Nt. BsmBI, Nb. BbvCI, Nt. BbvCI, or Nt.
BspQI.

61. The DNA molecule of claim 57, wherein the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease is Nt. BsmAI; Nt.
BtsCI; N. ALw1; N. BstNBI; N. BspD6I; Nb. Mva12691; Nb. BsrDI; Nt. BtsI; Nt.
BsaI; Nt.
Bpul0I; Nt. BsmBI, Nb. BbvCI, Nt. BbvCI, or Nt. BspQI.

62. The DNA molecule of any one of claim 31 to 59, wherein the nicking endonuclease that recognizes the first, second, third, and/or fourth restriction site for nicking endonuclease is a programmable nicking endonuclease.

63. The DNA molecule of claim 57, wherein the nicking endonuclease that recognizes the fifth and sixth restriction site for nicking endonuclease is a programmable nicking endonuclease.

64. The DNA molecule of claim 62 or 63, wherein the nicking endonuclease is a Cas nuclease.

65. The DNA molecule of any one of claim 31 to 64, wherein the expression cassette further comprises a promoter operatively linked to a transcription unit.

66. The DNA molecule of claim 65, wherein the transcription unit comprises an open reading frame.

67. The DNA molecule of claim 65 or 66, wherein the expression cassette further comprises a posttranscriptional regulatory element.

68. The DNA molecule of claim 65 or 66, wherein the expression cassette further comprises a polyadenylation and termination signal.

69. The DNA molecule of any one of claims 65 to 68, wherein the size of the expression cassette is at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb.

70. A kit for expressing a human GDE in vivo, the kit comprising 0.1 to 500 mg of a DNA molecule of any of claims 31 to 69 and a device for administering the DNA
molecule.

71. The kit of claim 70, wherein the device is an injection needle.

72. A composition comprising one or more DNA molecules of any of claims 31-69, and a pharmaceutically acceptable carrier.

73. The composition of claim 72, wherein the carrier comprises a transfection reagent, a nanoparticle, a hybridosome, or a liposome

74. The composition of claim 72 or 73 for use in medical therapy.

75. The use of a composition of any of claims 72 to 74 for preparing or manufacturing a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of GDE in a subject need thereof.